Cas9-Based Genome Editing for Disease Modeling and

Review pubs.acs.org/CR

CRISPR/Cas9-Based Genome Editing for Disease Modeling and Therapy: Challenges and Opportunities for Nonviral Delivery Hong-Xia Wang,† Mingqiang Li,† Ciaran M. Lee,‡ Syandan Chakraborty,† Hae-Won Kim,§ Gang Bao,‡ and Kam W. Leong*,† †

Department of Biomedical Engineering, Columbia University, New York, New York 10027, United States Department of Bioengineering, Rice University, Houston, Texas 77005, United States § Institute of Tissue Regeneration Engineering (ITREN) and Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 31116, Korea ‡

ABSTRACT: Genome editing offers promising solutions to genetic disorders by editing DNA sequences or modulating gene expression. The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) technology can be used to edit single or multiple genes in a wide variety of cell types and organisms in vitro and in vivo. Herein, we review the rapidly developing CRISPR/Cas9-based technologies for disease modeling and gene correction and recent progress toward Cas9/guide RNA (gRNA) delivery based on viral and nonviral vectors. We discuss the relative merits of delivering the genome editing elements in the form of DNA, mRNA, or protein, and the opportunities of combining viral delivery of a transgene encoding Cas9 with nonviral delivery of gRNA. We highlight the lessons learned from nonviral gene delivery in the past three decades and consider their applicability for CRISPR/Cas9 delivery. We also include a discussion of bioinformatics tools for gRNA design and chemical modifications of gRNA. Finally, we consider the extracellular and intracellular barriers to nonviral CRISPR/Cas9 delivery and propose strategies that may overcome these barriers to realize the clinical potential of CRISPR/Cas9-based genome editing.

CONTENTS 1. Introduction 2. Development, Mechanism of Action, and Advantages of CRISPR/Cas9 Systems 2.1. Discovery and Mechanism of Action of CRISPR/Cas9 Systems 2.2. Comparison of Alternative Nucleases with CRISPR/Cas9 Systems 2.2.1. Comparison of Meganucleases, ZFN, and TALEN with CRISPR-Associated Nucleases 2.2.2. Comparison of Different CRISPR Systems 2.3. Comparison of CRISPRi and RNAi 2.4. Bioinformatics Tools for sgRNA Design 2.4.1. Predicting sgRNA Activity Using in Silico Design Tools 2.4.2. In Silico Identification of CRISPR OffTarget Sites 2.4.3. Experimental Determination of CRISPR Off-Target Sites 3. Application of CRISPR/Cas9 Systems for Disease Modeling and Therapy 3.1. Disease Modeling 3.2. Correcting Monogenic Disorders 3.3. Correcting Nonmonogenetic Diseases

© 2017 American Chemical Society

3.4. Combating Infectious Diseases 4. Factors Influencing the Therapeutic Efficacy of Genome Editing 5. Current Delivery of CRISPR/Cas9: Pros and Cons of Viral versus Nonviral 5.1. Modes of CRISPR/Cas9 Delivery 5.1.1. Current Delivery of Cas9 Expression DNA 5.1.2. Current Delivery of Cas9 Expression mRNA 5.1.3. Current Delivery of Cas9 Protein 5.2. Viral versus Nonviral Delivery 6. Potential Strategies To Adapt Current Nonviral Delivery Systems for CRISPR/Cas9 Delivery 6.1. Potential Physical Approaches for CRISPR/ Cas9 Delivery 6.1.1. Microinjection 6.1.2. Electroporation 6.1.3. Hydrodynamic Injection 6.2. Potential Chemical Approaches for CRISPR/ Cas9 Delivery 6.2.1. Potential Vectors for Cas9 Expression Plasmid Delivery

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Received: November 29, 2016 Published: June 22, 2017 9874

DOI: 10.1021/acs.chemrev.6b00799 Chem. Rev. 2017, 117, 9874−9906

Chemical Reviews 6.2.2. Potential Vectors for Cas9 mRNA Delivery 6.2.3. Potential Vectors for Cas9 Protein Delivery 6.2.4. Combination of Viral Delivery for Cas9 Expressing Cassettes and Nonviral Delivery for sgRNAs 7. Critical Barriers to Nonviral Delivery of CRISPR/ Cas9 7.1. Extracellular and Intracellular Barriers of Nonviral Delivery of CRISPR/Cas9 7.2. Chemical Modifications To Overcome the Instability of sgRNA 7.3. Manufacturing Considerations for Nonviral Delivery of CRISPR/Cas9 8. Concluding Remarks Author Information Corresponding Author ORCID Notes Biographies Acknowledgments References

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editing is a more flexible platform due to its dependence on simpler base-pairing between an engineered single guide RNA (sgRNA) and the target DNA site adjacent to the protospacer adjacent motif (PAM) sequence, and the subsequent introduction of a double-strand break (DSB) on the genome by the Cas9 endonuclease.8 In the most commonly used Streptococcus pyogenes CRISPR/Cas9 (SpCas9) system, Cas9 is able to target DNA sequence that is upstream of 5′-NGG PAM sequences. CRISPR/Cas9 has minimal requirements for target design and straightforward construction of sgRNAs, enabling faster and easier implementation than any other genome editing strategies. As biomedical scientists continue to unravel the basic mechanisms of CRISPR/Cas9 and minimize the off-target effects, gene therapy researchers and biomedical engineers have begun to optimize delivery systems for these gene-editing elements.9 Most of the CRISPR/Cas9 studies so far have relied on either physical or viral delivery (Figure 1). Physical

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1. INTRODUCTION Gene therapy in the past decades has focused on restoration of missing gene function by viral or nonviral transgene expression.1 However, even after successful gene transfer, the disease-causing mutations still persist, which may lead to a dominant negative effect on the normal transgene. A preferred solution would be to correct the mutation. In addition, gene transfer resulting in random integration may disrupt endogenous genes and cause the emergence of new mutations. These drawbacks necessitate new strategies to correct diseasecausing genetic mutations. Genome editing can precisely disrupt, insert, or replace a DNA sequence at a specific locus in the genome, offering a powerful tool for biological research and therapy of genetic diseases.2 Traditionally, gene targeting has been achieved by using the host homologous recombination (HR) system to incorporate a homology sequence-flanked transgene.3 However, the low frequency of HR-based transgene integration events in the genome presents an enormous challenge for its robust application in large-scale gene editing. To overcome this limitation, a series of programmable nuclease-based genome editing technologies have been developed in recent years.2 Programmable site-specific nucleases are used to introduce sitespecific DNA breaks into the genome. Subsequent repair of the DNA break occurs by a homology directed repair (HDR)-based mechanism or via nonhomologous end joining (NHEJ), resulting in efficient genome editing. In recent years, programmable nucleases including meganucleases, zinc-finger nucleases (ZFNs), and transcription-activator-like effector nucleases (TALENs) have contributed immensely to the development of gene editing.4−6 However, these methods use proteins to recognize the target DNA sequence, and thus for each target, a new protein has to be engineered, a time- and labor-intensive process. In contrast, the recently developed site-specific gene editing platform, the clustered regularly interspaced short palindromic repeat (CRISPR) associated proteins 9 (CRISPR/ Cas9) technology, uses RNAs for site recognition.7 As compared to its predecessors, CRISPR/Cas9-based gene

Figure 1. Techniques for CRISPR/Cas9 delivery. ArgNPs, cationic arginine gold nanoparticles; CPP, cellular penetration peptide; i.j., injection; t.f., transfection; AAV, adeno-associated virus.

methods, such as electroporation and microinjection, have been successfully used;10−13 however, cell viability and difficulty to apply in vivo limit them to in vitro and ex vivo applications.14−16 Viral vectors such as lentivirus (LV), adenovirus (AV), and adeno-associated virus (AAV) can also be used to efficiently deliver the CRISPR/Cas9 system,17−20 but concerns of insertional mutagenesis, carcinogenesis, and immunogenicity associated with viral delivery still linger.21 Chemical methods of delivering the CRISPR/Cas9 system via nanocomplexes have the potential to address many of these limitations, particularly with respect to safety, large packaging capacity, easier synthesis, and pharmacological issues.21 Recently, researchers have used lipid-based nanoparticles,22−26 polyethylenimine (PEI),27 Ca3(PO4)2,28 FuGENE 6 transfection reagents,29 cell-penetrating peptides (CPP),30 7C1 9875


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Figure 2. Timeline of the discovery and development of CRISPR/Cas9 systems. CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR-associated protein 9; DSB, double-strand break.

nanoparticles,31 cationic arginine gold nanoparticles,32 “coreshell” artificial virus,33 and DNA nanoclews,34 to achieve CRISPR/Cas9 delivery in vitro and in vivo. Excellent reviews regarding CRISPR/Cas9 development have appeared in the past four years focusing mostly on the molecular biology and viral delivery.2,8,35−42 Herein, we review the development and application of the CRISPR/Cas9 system for disease modeling and therapy, and then describe possible modes for CRISPR/ Cas9 delivery. After comparing the pros and cons of viral versus nonviral delivery in molecular design and formulation, we offer a critical analysis of the barriers to nonviral gene transfer, ranging from extracellular to intracellular transport and biomanufacturing. Delivery will likely become the bottleneck in the eventual clinical translation of the CRISPR/Cas9 technology. This Review aims to highlight the lessons learned from nonviral gene delivery in the past three decades and to stimulate innovations that can help fulfill the clinical potential of CRISPR/Cas9-based genome editing.

(crRNAs) could serve as small gRNAs interfering with virus proliferation.7 The same year, Marraffini et al. reported the type III CRISPR/Cas system from Staphylococcus epidermis targeted DNA rather than RNA to mediate adaptive immunity.49 Following these initial studies, the basic function and mechanism of CRISPR systems were further defined in the next few years.8 In 2012, Martin et al. figured out the crRNA base-paired to trans-activating crRNA (tracrRNA) to form a two-RNA structure and directed the Cas9 to introduce DSB in target DNA.50 In 2013, Cong et al. and Mali et al. simultaneously published pioneering studies showing the application of engineered CRISPR systems to accomplish genome editing in mammalian cells for the first time.51,52 Subsequently, a flurry of papers showed the efficacy of this technology to edit eukaryotic genomes including that of zebrafish,53 mice,54 and monkey.55,56 In 2014, Yin et al. delivered CRISPR/Cas9 by hydrodynamic injection to mouse liver to correct the hereditary tyrosinemia type I, a fatal genetic disease, which is the first report of correcting disease in vivo by application of CRISPR/Cas9.57 Efforts are currently underway to apply CRISPR/Cas9 gene editing in the context of human disease therapy. Recently, the U.S. National Institutes of Health (NIH) approved a project to use CRISPR/Cas9-modified cells for the treatment of cancer, which will start at end of 2016.58 Also, a clinical trial approved by the West China Hospital’s review board has been initiated to test the utility of gene-edited cells in treating patients with lung cancer.59 The SpCas9 system is able to target DNA sequence that is upstream of the PAM sequence containing 5′-NGG-3′ via gRNA-directed recognition (Figure 3). It can be guided by two RNAs that form a duplex: the crRNA, which specifies the target, and the tracrRNA. These two RNAs can also be combined into a chimeric single-guide RNA (Figure 3A).50 Wild-type Cas9 is a multifunctional protein that has two nuclease domains, the RuvC- and HNH-like domains (Figure 3A). Wild-type Cas9 is a multifunctional protein that has two nuclease domains, the RuvC- and HNH-like domains (Figure 3A). The HNH-like domain cleaves the DNA strand complementary to the sgRNA, while the RuvC-like domain cleaves the other strand, thereby generating a DSB in the DNA. The introduction of point mutations into Cas9 nuclease domains, for example, D10A or/and H840A (Asp10 → Ala, His840 → Ala), in SpCas9 can generate a Cas9 nickase or a catalytically inactive Cas9 (dCas9) (Figure 3B and C). The wild-type Cas9 can introduce a DSB at the target site, which can be repaired via a NHEJ- or HDR-based mechanism

2. DEVELOPMENT, MECHANISM OF ACTION, AND ADVANTAGES OF CRISPR/Cas9 SYSTEMS 2.1. Discovery and Mechanism of Action of CRISPR/Cas9 Systems

The discovery and development of CRISPR/Cas9 systems are shown in Figure 2. The origins of CRISPR-related research can be traced back to 1987 when Nakata and co-workers reported a curious set of interspaced short repetitive sequences downstream of the E. coli iap gene.43 The significance of these repeats was not immediately clear. In the next 15 years, more such repeat elements were reported in other bacteria and archaea. This led to heightened interest and speculation regarding their possible functions. In 2002, Ruud et al. used the term CRISPR for the first time to describe this family of interspaced repeats.44 They identified the CRISPR-associated (Cas) genes, which were invariably located adjacent to a CRISPR locus. This close association indicated a functional relationship between the Cas genes and the CRISPR loci.44 In 2005, Mojica et al., Pourcel et al., and Bolotin et al. managed to trace the origin of the CRISPR spacer sequences to plasmids and phages.45−47 Relating previous reports of transcription from the CRISPR loci and Cas proteins containing nuclease and helicase domains, researchers proposed that CRISPR might be an adaptive defense system against phage infection. In 2007, Barrangou et al. provided the first experimental evidence of type II CRISPR/Cas system-mediated adaptive immunity.48 The next year, Brouns et al. proved that mature CRISPR RNAs 9876


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site in a template-dependent manner. Single-nuclease-domainmutated Cas9 nickases cleave a single strand to produce nicks (Figure 3B). Cas9 nickase-produced single nick is healed preferentially by HDR rather than NHEJ, even with a low HDR efficiency.60 Cas9 nickases used in conjunction with a pair of offset sgRNAs binding to opposite strands of the target site can create a staggered DSB (Figure 4B), which can be used to induce higher HDR frequencies as compared to Cas9 nickases and single sgRNAs.61 Using Cas9 nickases to cause paired nicking can increase the specificity of Cas9 by up to 1500-fold relative to wild-type Cas9.61 This is because the off-target nick sites can usually be flawlessly repaired aided by the uncut strand. Shen et al.62 and Cho et al.63 also confirmed that paired Cas9 nickases could efficiently reduce the off-target effect. Fu et al. reported that combining paired nickases with truncated gRNAs could further decrease undesired mutagenesis at some off-target sites without sacrificing on-target genome editing efficiencies.64 Catalytically inactive dead Cas9 (dCas9), which has mutations in both of the nuclease domains, cannot cleave DNA, but it can bind to the targeting site, sometimes silencing the gene expression (Figures 3B and 4C).65,66 dCas9 can mediate transcriptional repression or activation when fused to transcription modulating domains like SID, KRAB, and VP64.18,67−72 Extending the sgRNA sequence with stem loop domains that recruit RNA-binding proteins such as MS2 can also benefit the dCas9/sgRNA system to regulate the transcriptional fate.68,73,74 Mali et al. first showed that a sgRNA extended with MS2 hairpins could recruit activators VP64 (MS2-VP64) to a reporter gene and resulted in a 12-fold increase over dCas9 fused to VP64 (dCas9-VP64). 68 Combining the MS2-VP64 and the dCas9-VP64 provided an additional 1.3-fold increase in gene upregulation.73 Epigenetic

Figure 3. Mechanism of CRISPR/Cas9. (A) The wild-type Cas9 nuclease cleaves DNA to introduce a double-strand break via its RuvC and HNH nuclease domains. (B) Cas9 nickases with point mutations at HNH or RuvC domains can bind to the target site and only cleave a single strand of DNA. (C) The dCas9 variant can bind DNA but cannot cleave DNA because of RuvC and HNH nuclease domain mutation. sgRNA, single-guide RNA; PAM, protospacer adjacent motif.

(Figure 4A). The repair based on the NHEJ pathway is errorprone and often results in insertions and/or deletions (indels) at the site of the break. By providing a homologous sequence, subsequent repair of the break by HDR can replace the target

Figure 4. Applications of CRISPR/Cas9. (A) Wild-type Cas9 nuclease cleaves DNA to form a double-strand break, which can be repaired via a NHEJ-based or HDR-based mechanism and introduces an indel, insertion, or replacement into the genome. (B) Two Cas9 nickases with point mutations in the HNH (or RuvC) domains can bind to opposite strands of the target site and create a staggered double-strand break. (C) dCas9 can be used for gene silencing, activation, and modification as well as gene labeling/imaging. Cas9, CRISPR-associated protein 9; dCas9, dead Cas9; sgRNA, single-guide RNA; PAM, protospacer adjacent motif. 9877


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bases A, T, G, and C) in TALENs is easier than assembling ZFN subunits recognizing 64 possible DNA triplet combinations. Moreover, TALEN’s domains are independent of one another and can be quickly assembled, while ZFN motifs need to interact with the neighboring ones; this causes technical challenges associated with the linkage protein engineering. However, like ZFNs, it requires the two arms of TALENs to bind to the sequence with the correct orientation and appropriate spacing for the efficient dimerization of FokI nuclease and subsequent DNA cleavage.89 In the last four years, CRISPR/Cas9 technology has emerged as the preferred genome editing technology by obviating the need for reengineering the protein domains to target new sequences.8 The Cas9 protein can be retargeted to new sequences by simple alterations to the guide RNA sequence. Moreover, the CRISPR-based system offers multiplexing ability to simultaneously target multiple sequences for editing. Unlike other gene editing systems, CRISPR/Cas9 is also insensitive to the methylation status of the targeted area.60 However, the specificity of CRISPR/Cas9 system is one of the main concerns that needs to be addressed. In 2013, Hsu et al., Fu et al., and Cradick et al. reported the off-target effect of CRISPR/Cas9 system.60,97,98 To address this issue, several strategies have been reported: (1) reducing the amount of active Cas9 in the cell;60,99,100 (2) using two mutant Cas9 nickases to create a staggered double-strand break (Figure 3C);61,68 (3) truncating the guide RNA sequence with shorter regions of target complementarity 140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 nickases.77,78 In addition, dCas9 also can be fused to fluorescent proteins such as green fluorescent protein (GFP), for enabling live-cell imaging of chromosomal loci (Figure 4).69 Another alternative strategy for live-cell imaging and chromatin labeling based on dCas9 has been achieved by incorporating MS2 or PP7 RNA aptamers into the sgRNA, and then recruiting the corresponding MS2 or PP7 coat proteins fused with different fluorescent proteins to the target genomic loci.79 2.2. Comparison of Alternative Nucleases with CRISPR/Cas9 Systems

2.2.1. Comparison of Meganucleases, ZFN, and TALEN with CRISPR-Associated Nucleases. There are four major classes of nucleases used for genome engineering: meganucleases, 80−82 ZFNs,83−86 TALENs,87−90 and the CRISPR-associated nucleases such as Cas98 and Cpf1.91 The first three depend on recognition of specific sequences by programmable protein−DNA interactions, whereas CRISPR/ Cas9 and CRISPR/Cpf1 depend on the base pairing specificity of a short RNA guide molecule with the genomic DNA. Meganucleases are endonucleases with extended DNA recognition sites (>14 base pairs).81 Protein engineering of the recognition sites for the limited number of naturally occurring meganucleases has ensured the generation of multiple synthetic endonucleases with varying sequence specificity.81,92,93 However, the complexity in engineering these endonucleases and often their low editing efficiency have hindered the progress of this technology. The potential of targeted genome engineering was unlocked by the creation of the first ZFNs showing in vitro activity in 1996.83 Three to six zinc-finger DNA-binding domains (ZFDBDs) are fused in tandem along with a FokI endonuclease to assemble the ZFNs. The sequence specificity of ZFNs arises from individual ZFDBD recognizing a triplet nucleotide code. The placement of two ZFDBD arrays in close approximation allows for the FokI endonuclease to dimerize and consequently produce DSB in the DNA.5 ZFNs have recently fallen out of favor because of the technical challenges associated with the linkage engineering the protein domains, the preference of engineered ZFNs for Grich sequences, the need to have two synthetic zinc-finger DNA-binding proteins with appropriate orientation and spacing, and the presence of more robust alternatives.94 Like ZFNs, TALENs are comprised of a tailor-made DNArecognition domain fused to a nonspecific FokI nuclease domain.89 A typical TALE DNA-binding domain recognizes 14−20 bases with a conserved thymine (T) base immediately preceding its 5′ boundary. Highly conserved repeating subunits recognize individual bases. Therefore, extensive cloning effort is needed to assemble a customized TALEN molecule.95 These repeats also leave the construct vulnerable to recombination, especially the ones cloned into a lentiviral vector.96 The modular assembly of four different subunits (to recognize the 9878


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Figure 5. Performance of CRISPR/Cas9 prediction algorithms. (A) Overview of CRISPR library screening for enrichment of highly active gRNAs. (B) Activity of 92 gRNAs designed with tools that predict highly active gRNAs (gray) or with no guidelines (red). No improvement in the frequency of highly active gRNAs is seen with the incorporation of current prediction guidelines. Previously unpublished data. (C) Performance of sgRNA design tools with data sets from independent studies. Data summarized from ref 121 with the permission of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). Copyright 2016.

overhangs facilitated by the more efficient NHEJ repair process. Another important distinguishing feature of Cpf1 is its cleavage at a site distal to the PAM and far from the seed sequence, which potentially allows multiple cycles of NHEJ repair without disrupting the essential target sequence. By contrast, even a single cycle of NHEJ repair in Cas9’s cleavage site may result in target site disruption. This attribute of Cpf1 provides opportunities for multiple cycles of cleavage at the site that has been repaired and restored to the original unedited sequence.

Cas systems (Cas9), as well as the recently characterized type V-A (Cpf1).91 Because Class 2 CRISPR systems require only a single protein for genome editing, they have the potential for widespread utilization due to their simple design and ability to precisely target multiple sites in the genome at the same time. Shmakov et al. used a computational approach to extract three distinct Class 2 CRISPR systems different from Cas9: C2c1 and C2c3 (type V-B and type V-C, respectively), and C2c2 (type VI).113 Recently, C2c2 was shown to be a single-RNA guided Rnase, demonstrating for the first time that some Class 2 systems can target RNA.114 Zetsche et al. described the use of Cpf1, the effector protein of type V Class 2 CRISPR system, for genome engineering.91 CRISPR/Cpf1 systems from two species (Acidaminococcus and Lachnospiraceae) were adapted in a mammalian expression system to demonstrate efficient genome editing activity in human cells. Cpf1 differs from the well-characterized Cas9 molecule in several ways, including the presence of only the RuvC-like endonuclease domain and the absence of HNH endonuclease domain, the requirement of a single RNA for target recognition rather than needing both tracrRNA and crRNA, and the generation of staggered 5′ overhang cuts in the target DNA rather than blunt end cuts generated by Cas9.91 Short guide RNAs (42 nt) required for Cpf1 targeting can be easily produced and are less expensive than the 110 nt guide RNA needed for Cas9 through chemical synthesis. Recognition of T-rich PAMs by the Cpf1-family also distinguishes it from Cas9’s preference for G-rich PAMs, thereby expanding the toolbox for genome editing. This feature of Cpf1 allows the editing of hitherto difficult-to-edit AT-rich areas of the genome as well as genomes with relative abundance of A and T.91 The introduction of HDR is an inefficient process, even more so in cells not dividing. In this respect, the sticky ends in the genomic DNA resulting from Cpf1 staggered cuts may be helpful to introduce genes with complementary sequence

2.3. Comparison of CRISPRi and RNAi

Both the CRISPR/Cas9 and the RNAi technologies can be used to decrease a target gene expression, and both of them depend on a critical short RNA sequence for specific locustargeting. However, there are distinct differences between them: (1) CRISPR/Cas9 also can be used for gene insertion, mutation, and activation, whereas RNAi is mostly restricted to knocking down gene expression. (2) The gene deletion mediated by wild-type Cas9 is permanent and complete, but the gene-interfering effect by RNAi is transient and partial. Thus, for decreasing gene expression, CRISPR/Cas9-based therapeutics is advantageous over RNAi because it does not require repetitive administration. (3) Whereas RNAi via shRNA may induce a nonspecific gene regulation by microRNA pathways via saturation of DICER and DGCR8,115 the lack of endogenous CRISPR/Cas9 system in eukaryotes obviates this concern. However, this is also a “double-edged sword”, as siRNA can use the endogenous protein machinery of microRNA systems and only a small RNA needs to be delivered or expressed. (4) In contrast with the delivery of RNAi-based therapeutics, the delivery of CRISPR/Cas9-based therapeutics by both viral and nonviral means presents a greater challenge because the canonical SpCas9 expression DNA (typically around 8 kb), mRNA, or protein are all large molecules. Overall, CRISPR/Cas9 technology offers a broad 9879


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Figure 6. (A) Comparison of in silico tools based on 343 off-target sites identified by GUIDE-Seq unpublished data. (B) Experimental methods that have been developed to identify the genome-wide activity of CRISPR/Cas9 systems. (C) Overlap of off-target sites identified by GUIDE-Seq when carried out by two independent groups. SpCas9 and SaCas9 sgRNAs both target VEGFA. Data summarized from refs 139 and 140. Reference 139, Copyright 2015 Nature Publishing Group. Reference 140, under the permission of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/). Copyright 2015.

correlating the predicted score with observed activity in zebrafish and Xenopus laevis embryos.118 However, the ability of these algorithms to predict highly active sgRNAs in human cells has not yet been established. A direct comparison of sgRNA designs using available prediction algorithms and with randomly designed sgRNAs yielded no difference in the ratio of highly active sgRNAs (Figure 5B), demonstrating the deficiency of the existing sgRNA design tools. Therefore, even with the use of CRISPR design tools for activity prediction, multiple sgRNAs are still needed experimentally to identify a highly active sgRNA. Furthermore, a recent comparative analysis of several sgRNA design tools found evidence of algorithmic overfitting in the original studies.121 This study demonstrated that the Spearman correlation of the top performing design tools dropped significantly when tested against data sets not used for algorithm training in the original studies (Figure 5C). Despite limitations in designing sgRNAs for a defined genomic locus, these algorithms could prove useful for determining sgRNA sequences for large CRISPR libraries where multiple sgRNAs are designed for each target gene thus introducing a buffer of redundancy against poor sgRNAs. However, further refinement is required to improve our ability to predict the activity of a particular sgRNA sequence. 2.4.2. In Silico Identification of CRISPR Off-Target Sites. A major challenge in using CRISPR/Cas9 for genome engineering is the high incidence of DNA cleavage at off-target sites. Because the target specificity of a gRNA relies on

applicability that cannot be matched by RNAi. On the other hand, its delivery challenge is significantly higher because of its size and the site of action is in the nucleus, whereas RNAi is active in the cytoplasm. 2.4. Bioinformatics Tools for sgRNA Design

2.4.1. Predicting sgRNA Activity Using in Silico Design Tools. For targeting a specific genomic locus, not all sgRNAs designed are equally active, and some may fail to work. There are several online search tools that have been developed to predict the activity of a sgRNA design in silico. The advantage of using such a search tool would be a reduction in the experimental workload required to identify sgRNAs with higher activity. The algorithms underlying these tools are based on library screening (sgRNA Designer116 or sgRNA Scorer117), sgRNA activity in zebrafish embryos (CRISPRScan118), refinement via sequence determinants (Sequence Scan for CRISPR119), or sgRNA secondary structure and self-folding properties (WU-CRISPR120). Algorithms developed on the basis of library screens have the advantage of using a large data set from different cell types (Figure 5A). However, only a weak correlation is found when both sgRNA Designer and sgRNA Scorer are tested with the same data set.117 Although these two algorithms were validated with a test data set from CRISPR library screens, neither was tested with sgRNAs predicted to be highly active. The CRISPRScan algorithm was built upon data from 1280 sgRNAs tested in zebrafish embryos, and validated by using additional sgRNAs selected by the algorithm and 9880


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methods use different strategies to identify CRISPR/Cas9induced DSB via catalytically dCas9 binding,138,141,142 DSB capture of integrase-defective lentiviral vectors,137 doublestranded oligonucleotide (dsODN),122 or translocations,143 in vitro DNA cleavage and whole genome sequencing,136 and in situ DSB labeling.20,144 Cas9 ChIP assays use a catalytically dead version of Cas9 (dCas9), which retains its DNA binding ability, to determine the genome-wide binding profile of a particular gRNA complexed with dCas9. ChIP-seq can easily identify the on-target site and hundreds of genome-wide offtarget binding sites for several sgRNAs.138,141,142 However, in one of these studies, out of 295 identified dCas9 binding sites, 1 had off-target activity above background.142 These studies have proposed that while the seed region alone can facilitate Cas9 binding, more extensive gRNA-DNA hybridization is required to successfully cleave the DNA target site. In its native state, the HNH nuclease domain of Cas9 is not situated close enough to interact with the target DNA for cleavage. A conformational change in the HNH nuclease domain is required for robust DNA cleavage.145 This conformational change is maximal with full complementarity between the sgRNA and the DNA target sequence. Truncated sgRNAs often result in reduced conformational changes in the HNH domain, and it is likely the requirement of a full-length sgRNA-DNA hybrid (including mismatches) for DNA cleavage is the reason why the large number of Cas9 bound sites identified by ChIP-Seq experiments exhibit no detectable DNA cleavage.146 The digenomeseq assay identifies off-target sites via in vitro digestion of genomic DNA and whole genome sequencing.136 Only a subset of potential off-target loci identified via this method was found to have significant cleavage activity when assayed by next generation sequencing. This discrepancy suggests that accessibility of the target locus is important and that certain off-target events may be cell-type specific. In identifying CRISPR/Cas9 off-target sites, the capture of DNA breaks in situ has proved more successful than in vitrobased approaches. The GUIDE-seq method uses a short dsODN to tag DSBs.122 As shown in a recent study, a large number of off-target sites were found for 10 full-length sgRNAs and 3 truncated sgRNAs. The number of off-target sites found per sgRNA ranged from 0 to 151, demonstrating that the GUIDE-seq method has the potential to be a powerful tool capable of identifying genome-wide CRISPR off-target sites without any prior knowledge of or bias toward these sites. However, recent results suggest that further refinement of the method is necessary to standardize readouts from off-target screening. For example, using the GUIDE-seq method, two different sets of off-target sites were found independently by two groups for the same sgRNA.122,140 While there is considerable overlap between them, the two sets are not identical, and 18 and 33 off-target sites were missed respectively by the two sets (Figure 6C). When the off-target profile of a Staphylococcus aureus Cas9 (SaCas9) sgRNA was analyzed by both groups using GUIDE-seq, there was very little overlap between the off-target sites identified (Figure 6C), possibly due to the use of different sgRNA lengths.139,140 To date, the GUIDE-Seq method has been successfully applied to CRISPR/ Cas9 off-target site identification with a small number of cell types, and it remains to be seen whether it would work in primary cells such as hematopoietic stem cells. One alternative is to remove the reliance on tag integration and directly detect DNA DSBs, allowing the capture of DNA breaks in virtually any cell type. One such method is BLESS (direct in situ breaks

Watson−Crick base pairing, a sgRNA can hybridize to DNA sequences containing base mismatches relative to the intended target, resulting in off-target cleavage.60,97,98 CRISPR/Cas9 offtarget sites tend to contain a minimal number of mismatches. However, DNA sequences with up to 6 mismatches122 have been reported, and off-target sites that contain a DNA or RNA bulge in addition to base mismatches have also been identified.123 Off-target cleavage can induce detrimental changes to the genome, including mutation, insertion, deletion, inversion, and translocation. Given that CRISPR/Cas9 systems could have a high degree of off-target activity at closely matched sites,60,97,98 many web-based search tools exist for identifying potential off-target sites with algorithms heavily based on sequence composition,60,124−132 except for a few tools that allow for the inclusion of DNA or RNA bulges.133,134 However, all tools developed to date have restrictions in either the number of mismatches, the inclusion of bulges, or the allowable PAM sequences, thus prohibiting the identification of all potential off-target sites (Figure 6A). The CROP-IT tool incorporates data from ChIP-seq assays that map dCas9-gRNA binding profiles and common DNaseI accessible sites. Although this tool can identify a greater percentage of true off-target sites (80% of experimentally determined off-target sites),122 it also predicts a lot more potential off-target sites, with only 0.07% of these sites validated. Most of these online tools for the prediction of CRISPR off-target sites60,125,133,135 rely heavily on sequence homology between the sgRNA and the target genome, which often results in an inaccurate ranking of potential off-target sites, and presents the user with a dilemma. For example, the 320 sites identified by CROP-IT, a secondgeneration off-target search tool, represent only a small percentage of the 228 818 sites considered. Clearly, experimental validation of these sites would be difficult, highlighting the need for further improvement of current search algorithms. As off-target identification undergoes refinement, off-target search tools are useful for developing a nuclease design, synthesis, and analysis pipeline. For example, the CRISPRScan prediction algorithm has a custom genome browser track containing all possible gRNA designs for six common species, whereas off-target algorithms identify closely matched sites of potential gRNAs, and those tools not containing a repeat masker can identify sgRNAs that have multiple perfectly matched sites or off-target sites that fall within repetitive DNA elements. There are several hurdles to establishing a common set of in silico design rules for searching and ranking CRISPR off-target sites, which may be overcome by gaining a better understanding of the molecular mechanisms of Cas9 binding and gRNA loading, the affinity between sgRNA and DNA target, and target accessibility. This is possible as more true offtarget sites are identified using genome-wide unbiased methods for identifying nuclease off-target sites (discussed below),122,136−138 and with the development of a database of all true off-target sites of CRISPR/Cas9 systems. To this end, the recent CRISPOR web tool has collected all known experimentally confirmed off-target sites in an effort to improve off-target prediction and ranking.121 Despite current limitations, in silico prediction of gRNA activity and specificity is a quick, cost-effective means to prescreen candidate gRNAs and facilitates the analysis of CRISPR genotoxicity. 2.4.3. Experimental Determination of CRISPR OffTarget Sites. Several new experimental methods have been developed to identify the genome-wide activity of CRISPR/ Cas9 systems in an “unbiased” manner (Figure 6B). These 9881


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Table 1. Examples of Application of CRISPR/Cas9 Systems for Disease Therapya disease type Monogenic Disorders cataracts

cell or organism

delivery mode

CRISPR/Cas9 mode

ref 10 11 157 12 158−160

19 20 161 162

mouse zygote mouse SSCs mouse/human ISC mouse germ line mouse muscle tissue

microinjections electroporation Lipofectamine microinjections AAV

mouse liver mouse liver

hydrodynamic injection lipid nanoparticles and AAV

Cas9 mRNA and sgRNA (Cas9 and sgRNA) expression DNA (Cas9 and sgRNA) expression DNA (Cas9 and sgRNA) expression DNA Cas9 expression DNA and sgRNA expression DNA (Cas9 and sgRNA) expression DNA Cas9 mRNA and sgRNA expression DNA

mouse liver mouse liver cancer cells cancer cells

adenovirus AAV Lipofectamine jetPRIME/Fugene6

(Cas9 (Cas9 (Cas9 (Cas9

HIV-1 provirus-integrated human cells microglial/promonocytic/T cells iPSC

Lipofectamine/Ca3(PO4)2

28

lentivirus AAV

HBV

human T-lymphoid cells mouse spleen, liver, heart, lung, and kidney HepG2/HepG2.2.15 mouse liver

Cas9 expression DNA and sgRNA expression DNA (Cas9 and sgRNA) expression DNA Cas9 expression DNA and sgRNA expression DNA (Cas9 and sgRNA) expression DNA (Cas9 and sgRNA) expression DNA

Huh7/HepG2.2.15 Huh-7.5

HPV HSV-1 EPV

HeLa 293T/SiHa cells L7/TC260 cells human lymphoma cell

Ca3(PO4)2/Fugene6/Lipofectamine Lipofectamine/lentiviral electroporation

Cas9 expression DNA and sgRNA expression DNA (Cas9 and sgRNA) expression DNA Cas9 expression DNA and sgRNA expression DNA (Cas9 and sgRNA) expression DNA (Cas9 and sgRNA) expression DNA (Cas9 and sgRNA) expression DNA

27

HCV

PEI/Lipofectamine hydrodynamic injection Lipofectamine Lipofectamine

cystic fibrosis DMD

hereditary tyrosinemia Nonmonogenetic hypercholesterolaemia bladder cancer colon cancer Infectious Diseases HIV

Lipofectamine electroporation

and and and and

sgRNA) sgRNA) sgRNA) sgRNA)

expression expression expression expression

DNA DNA DNA DNA

57 25

125 13 163 164

165 166 29 167 168

a

SSC, spermatogonial stem cells; ISC, intestinal stem cells; DMD, Duchenne muscular dystrophy; AAV, adeno-associated viruses; HIV, human immunodeficiency virus; HBV, human hepatitis B virus; HCV, hepatitis C virus; HPV, human papillomaviruses; HSV-1, Herpes simplex virus type 1; EPV, Epstein−Barr virus; PEI, polyethylenimine.

by hydrodynamic injecting the Cas9 expression plasmid and sgRNAs to target wild-type tumor repressor genes Pten and p53 in murine liver.147 The induced mutations led to rapid development of liver cancer. An efficient method of applying CRISPR/Cas9 to induce several types of genomic rearrangements implicated as driver events in lung cancer including the CD74-ROS1, EML4-ALK, and KIF5B-RET gene fusions has been reported.148 This provides a convenient approach to study various types of lung cancers. As a primary tumor is often driven by multiple genes, editing one gene at a time may not be enough for establishing a highly complicated polygenic tumor model. CRISPR/Cas9 can edit multiple targets at the same time by codelivering multiple sgRNAs. This feature makes it a versatile tool for generating a tumor model with complexity similar to that occurring in human. Matano et al. used the CRISPR/Cas9 genome-editing system to introduce multiple driver gene mutations into the human colonic epithelium to model colorectal cancer.149 Zuckermann et al. demonstrated the utility of CRISPR/Cas9 genome-editing approach by deleting single (Ptch1) or multiple genes (Trp53, Pten, Nf1) in the mouse brain, resulting in the development of medulloblastoma and glioblastoma, respectively.150 Heckl et al. used the CRISPR/Cas9 system to induce multiple mutations in epigenetic modifiers, transcription factors, and cytokine signaling genes in mouse hematopoietic stem cells to generate a model of acute myeloid leukemia.151 CRISPR/Cas9 also shows its potential for generating other diseases models. Recent

labeling, enrichment on streptavidin, and next generation sequencing),144 which can detect DNA breaks by labeling free DNA ends, with the potential to facilitate genome-wide dynamic studies of CRISPR/Cas9 activity at on- and off-target sites. However, it cannot detect off-target sites that have undergone NHEJ repair and indel formation. A comprehensive comparison of current experimental methods for genome-wide CRISPR off-target detection is needed to establish their applicability, albeit the challenge is having overlapping sgRNAs in the study.

3. APPLICATION OF CRISPR/Cas9 SYSTEMS FOR DISEASE MODELING AND THERAPY Given the intense interest in CRISPR/Cas9 research, this technology is destined to enter the therapeutic domain. Some recent proof-of-principle studies have described successful deployment of this technology to establish in vitro and in vivo disease models or to correct genetic defects (Table 1). 3.1. Disease Modeling

Cancer is caused by complicated mechanisms involving a variety of genetic alterations of tumor suppressor genes and oncogenes. Developing suitable cancer models is quite useful for exploring the mechanisms of the initiation and progression of cancer, and for anticancer drug screening. Recent studies have shown the power of combining the CRISPR/Cas9 system with murine cancer models. A liver cancer model was generated 9882


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modification, it is tempting to apply this technology to treat nonmonogenic cardiovascular diseases, tumors, Alzheimer disease, and metabolic disorders (Table 1). One example of such use involves the knockdown of Pcsk9 (proprotein convertase subtilisin/kexin type 9) gene.19,20 Loss-of-function of this gene led to a decrease in plasma low-density lipoprotein (LDL) level, thereby showing therapeutic potential for hypercholesterolaemia. Recently, Ran et al. developed smaller Cas9 orthologues from Staphylococcus aureus (SaCas9) and targeted the Pcsk9 in the mouse liver by using a single AAV vector.20 Cancer therapy based on CRISPR/Cas9 technology has also been explored. In one study, sgRNAs and Cas9 controlled by two bladder cancer-specific promoters mutated the LacI gene, thereby re-expressing several normal phenotypespecific genes, which can be used to inhibit bladder cancer cell growth, induce apoptosis, and decrease cell motility.161 Antal et al. showed the correction of a loss-of-function PKC (Protein Kinase C) mutation by CRISPR/Cas9-mediated genome editing in a patient-derived colon cancer cell line and reduced tumor growth in a xenograft model.162

reports described the generation of mouse cardiomyopathy and heart failure models by delivering Myh6 sgRNA to cardiacspecific Cas9 transgenic mice. This method provides a strategy to rapidly editing genes in the heart.152 In addition to mouse models, CRISPR/Cas9 also facilitates the generation of rat,60,153 pig,154 and monkey55 models better suited for pharmacological studies and disease modeling. The induced pluripotent stem cells (iPSCs) with their unlimited self-renewal capability and ability to differentiate into cells of any lineage attract much attention for their value in disease modeling. In 2013, Horri et al. generated an iPSC model for immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF) syndrome using the CRISPR system.155 Their data suggest that the CRISPR system is highly efficient and useful for genome engineering of human hPSCs. In another study, Paquet et al. described a CRISPR/ Cas9-based genome-editing framework to selectively introduce mono- and biallelic sequence changes to hPSCs and generated a cell model with Alzheimer’s disease-causing mutations in amyloid precursor protein and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes.156

3.4. Combating Infectious Diseases

As the CRISPR/Cas9 system was initially identified as an antiviral adaptive immune system in bacteria, it can also be used as a novel technology to counter viral infections in humans (Table 1). Ebina et al. applied CRISPR/Cas9 technology in vitro as an anti-HIV therapy to mutate a long terminal repeat (LTR) sequence in proviral DNA of human immunodeficiency virus 1 (HIV-1).28 Recently, another study used CRISPR/Cas9 to mutate HIV-1 LTR U3 region and showed similar results.125 CCR5 is the major coreceptor used by HIV-1 to infect host cells. Humans with the CCR5Δ32 mutation are either resistant to HIV infections or have a slower progression of the disease. Ye et al. generated iPSCs homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of iPSCs using a combination of TALENs or CRISPR/Cas9 with piggy Bac technology.13 Hepatitis B virus (HBV)-mediated chronic hepatitis is one of the most common infectious diseases worldwide. HBVs covalently closed circular DNAs (cccDNAs) residing inside the infected cell is hard to clear by current therapeutics. Several in vitro and in vivo studies showed the introduction of HBV cccDNA-targeting CRISPR/Cas9 facilitated cleavage of HBV genome and aided in its cellular clearance.27,165 This RNA-guided endonuclease also provides a therapeutic strategy to cure human papillomaviruses (HPV),29 Herpes simplex virus type 1 (HSV-1),167 and Epstein−Barr virus (EBV).168 The CRISPR/Cas9 system was initially identified as an RNA-guided DNA endonuclease for sequence-specific dsDNA cleavage. However, the recent discoveries indicate that Cas9 could also bind and cleave RNA.169,170 Interestingly, Weiss et al. found the Cas9 endonuclease from Francisella novicida (FnCas9) was capable of targeting endogenous RNA171 and could be used for the inhibition of hepatitis C (HCV) virus.166

3.2. Correcting Monogenic Disorders

Monogenic disorders result from defects in a single gene in the human DNA. CRISPR/Cas9-based gene therapy holds great promise in correcting monogenic diseases (Table 1). Here, we show that mice with a dominant mutation in Crygc gene that causes cataracts could be rescued by coinjection into zygotes of Cas9 mRNA and a single-guide RNA (sgRNA) targeting the mutant allele. Monogenetic cataract disease in mice has been rescued by coinjection of Cas9 mRNA and sgRNA targeting the mutant allele into mouse zygotes.10 The mice were able to transmit the corrected Crygc allele to their progeny. In a subsequent publication, the same group corrected mutant Crygc in mouse spermatogonial stem cells (SSCs) with CRISPR/Cas9.11 They showed high rescue efficiency, without any evidence of offtarget effects. In another study, Schwank et al. used CRISPR/ Cas9 to correct a genetic defect associated with cystic fibrosis in human stem cells by editing the CFTR gene.157 Germ line CRISPR/Cas9-based repair of dystrophin gene (Dmd) mutation has been used to treat Duchenne muscular dystrophy (DMD), an inherited X-linked disease caused by mutations in the gene encoding dystrophin (a protein required for muscle fiber integrity).12 This procedure led to mdx mice carrying the corrected version of the mutant gene in 2−100% of the somatic cells. Correction of the mutant phenotype in adult mammalian organ was first reported when Yin et al. delivered CRISPR/ Cas9 by hydrodynamic injection to the mouse liver, resulting in expression of the wild-type Fah protein in ∼1/250 liver cells.57 The Fah-positive hepatocytes rescued the weight loss phenotype in that model. Recently, the same group increased the Fah correction efficiency to >6% of hepatocytes by tail vein injection of lipid nanoparticles carrying Cas9 mRNA in conjunction with AAV encoding a sgRNA and a repair template to induce repair in mouse liver.25 By the end of 2015, three groups simultaneously published similar studies on improving muscle function in a mouse model of DMD using AAVmediated CRISPR/Cas9 genome editing.158−160

4. FACTORS INFLUENCING THE THERAPEUTIC EFFICACY OF GENOME EDITING Although the CRISPR/Cas9 technology has built a number of disease models and corrected genes related to various diseases, numerous challenges still lie ahead for translation of this technology. Key factors influencing the therapeutic outcome of gene editing by the CRISPR/Cas9 system need to be recognized and effective strategies devised. The first obstacle

3.3. Correcting Nonmonogenetic Diseases

Given that CRISPR/Cas9 is a versatile editing tool for gene deletion, insertion, activation, repression, and even epigenetic 9883


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Figure 7. Factors interfering with the therapeutic efficacy for genome editing. (A) Ex vivo versus in vivo editing therapy. The edited cells are shown in pink and unedited cells are shown in blue. (B) Fitness of edited cells and therapeutic “threshold”. Adapted from ref 2. Copyright 2015 Nature Publishing Group.

Figure 8. Challenges of delivering different configurations of Cas9/gRNA elements by nonviral vectors.

should be the selection of an appropriate nuclease platform and proper design of sgRNAs, which has been reviewed above. The second obstacle is ensuring efficient delivery of the editing tools into the target cells in vivo or ex vivo (Figure 7A). In principle, both viral and nonviral vectors can be used for Cas9 expression via DNA/mRNA/protein as well as sgRNAs delivery, which will be discussed in detail in the following sections. The third

obstacle is efficient genome editing after the editing elements have entered the nucleus. The efficiency of NHEJ- and HDRmediated DSB repair varies substantially within cell types and cell states. Methods for improving the efficiency of HDR or NHEJ events as well as for reducing off-target effects will be important thrust areas for improving the overall therapeutic outcome (reviewed in REFS8,36,172). NHEJ produces small 9884


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Figure 9. Multiplex genome editing based on Cas9 expression DNA delivery. (A) SpCas9 can facilitate multiplex genome modification by using a crRNA array that contains two spacers targeting EMX1 and PVALB. Scheme shows the design of the crRNA array (upper). Both spacers mediate efficient protospacer cleavage (lower). Reprinted with permission from ref 51. Copyright 2013 The American Association for the Advancement of Science. (B) Schematic illustration of the design for multiplex genome editing in mouse embryonic stem cells (ESCs) (upper) and restriction fragment length polymorphism (RFLP) analysis of triple-gene-alleles mutation (lower). Reprinted with permission from ref 54. Copyright 2013 Elsevier Inc. (C) Genomic cleavage analysis of the all-in-one plasmid expressing seven sgRNAs targeting seven different genomic loci and the Cas9 nuclease. The blue and green bent arrows indicate the U6 and CBh promoters, respectively. The products from untransfected control cells (C means control group) and cells transfected with CRISPR/Cas9-nuclease vectors targeting seven (7) and single (1) loci were analyzed by agarose gel electrophoresis. Reprinted with permission from ref 180. Copyright 2014 Nature Publishing Group.

edited cells lack fitness as compared to the unedited ones. This problem can be partly solved by ex vivo genome editing, where the edited cells, possibly after expansion, can be reinfused into the patients (Figure 7A). However, the reinfused cells may also fail due to a lack of engraftment (Figure 7B). The last potential obstacle is immune response stimulated by Cas9, a foreign protein of bacterial origin, which has been verified.173,174 With the above factors in consideration, this Review will now present an overview of the current CRISPR/Cas9 delivery systems and suggest a roadmap to improve their efficiency with lessons learned from other gene delivery studies.

insertions or deletions at the cleavage site, whereas HDR needs a DNA template to replace the targeted allele by recombination with an alternative sequence. HDR templates usually are singlestranded oligonucleotides or plasmids containing alleles of target-adjacent sites, and they also need to be delivered into the cell nucleus. The delivery of HDR templates has been achieved with viral or nonviral vectors.25,52 Theoretically, HDR template delivery based on nanocarriers can follow the same principles as for other oligonucleotides or plasmid DNA delivery. This would entail formulation by electrostatic interactions, maximization of cellular uptake, protection from degradation, and translocation into cell nucleus. These requirements are similar for Cas9 plasmid/protein as well as sgRNAs delivery. Thus, we speculate that the design of nanocarriers for HDR templates can be conveniently combined with that for Cas9 plasmid/ protein or sgRNA delivery, which also facilitates the codelivery of Cas9 elements/sgRNAs and HDR templates to increase the HDR efficiency. The fourth obstacle influencing the therapeutic outcome is the health of the edited cells and the therapeutic threshold of editing (Figure 7B).2 Edited cells having a better ability to thrive as compared to the unedited cells, that is, display a higher fitness relative to unedited cells, help the edited gene product to achieve the therapeutic threshold necessary for a successful treatment outcome (Figure 7B). The therapeutic outcome is bleak in the case of inefficient gene editing or if the

5. CURRENT DELIVERY OF CRISPR/Cas9: PROS AND CONS OF VIRAL VERSUS NONVIRAL 5.1. Modes of CRISPR/Cas9 Delivery

5.1.1. Current Delivery of Cas9 Expression DNA. Two critical components (Cas9 nucleases and sgRNAs) are needed for CRISPR/Cas9 functional activity. Three nonviral platforms are available for delivering the Cas9 nucleases: Cas9 Expression DNA, Cas9 expression mRNA, and Cas9 protein (Figure 8). The most common-used SpCas9 is a large nuclease, whose expression cassette is longer than 4 kb and total plasmid size is usually bigger than 7 kb with the addition of plasmid backbone and other expression assistant elements.52,67,175 Sometimes 9885


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Figure 10. Genome editing based on Cas9 mRNA delivery. (A) (1) Outline of one-step correction of a genetic defect in cataract mouse model via the use of microinjection of Cas9 mRNA and sgRNA to the zygote. (2) A repaired mouse carrying the correct allele induced by HDR and a control heterogeneous mutant (Crygc+/−) mouse. The repaired mouse after CRISPR/Cas9-mediated gene correction is free of cataracts, as measured by the appearance of black lenses as compared to the opacity of the lens in control heterogeneous mutant mouse (Crygc+/−). (3) Histological analysis of lenses prepared from repaired mice via HDR and control heterogeneous mutant mice (Crygc+/−). While the heterogeneous mutant mouse (Crygc+/−) shows vacuole-like degeneration in the equatorial region of the entire eye, the cataract-free mouse exhibits normal histological features. Reprinted with permission from ref 10. Copyright 2013 Elsevier Inc. (B) (1) Outline of an efficient approach for generation genome modified cynomolgus monkeys by microinjection of Cas9 mRNA and sgRNA to the one-cell-stage embryos and achievement of Ppar-g and Rag1 double mutation in one step. (2) Sequences of modified Ppar-γ and Rag1 loci detected in founder cynomolgus monkeys. At least 18 TA clones of the PCR products were analyzed by DNA sequencing. N/N indicates positive colonies out of total sequenced. The PAM sequences are underlined and highlighted in green; the targeting sequences in red; the mutations in blue, lowercase; deletions (−) and insertions (+). Reprinted with permission from ref 55. Copyright 2014 Elsevier Inc. (C) In vivo therapeutic genome editing by combined AAV and nonviral delivery of Cas9 to correct Fah mutation. (1) Fahmut/mut mice were injected with AAV with sgRNA/HDR template and nanoparticles with Cas9 mRNA at indicated time points. These mice can be treated with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), an inhibitor of an enzyme upstream of Fah, to prevent toxin accumulation in hepatocytes. (2) Fah immunohistochemistry (IHC) in the Fahmut/mut mice liver shows the Fah expression was repaired by the treatment. (3) Fah+ positive cells were counted to determine the percentage. Reprinted with permission from ref 25. Copyright 2016 Nature Publishing Group.

inclusion of reporter genes would further increase its size.18,175,176 As compared to the Cas9 mRNA/protein, the Cas9 expression DNA should be more stable and cost-effective, but a longer lag time before Cas9 expression and a longer persistence of the transgene product may be disadvantageous. The persistence of Cas9 may lead to higher off-target effects.

Another obstacle for Cas9 expression DNA application is the requirement of nuclear entry of DNA (Figure 8). Plasmid DNA application is plagued by random integration of all or part of the plasmid DNA into the host genome. It should be noted that insertion of plasmid DNA sequences at off-target sites is difficult to detect. Even more problematic is that these foreign 9886


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sequences may cause host immune responses.150,177 Last, DNA transfection is often stressful to cells. For example, plasmid DNA introduced into cells may trigger cyclic GMP-AMP synthase activation.178 Along with the Cas9 expression cassette, the DNA sequence for gRNA expression can also be packed into the same expression cassette or built into separate expression cassettes. One of the significant advantages of the CRISPR/Cas9 system is to realize multiplexed genome editing by using multiple gRNAs targeting different genomic loci with a common Cas9 expression cassette.51,69,179 Cong et al. first reported that editing of several sites within the mammalian genome could be achieved by encoding multiple guide sequences into a single CRISPR plasmid and transfected by Invitrogen Lipofectamine 2000 (Figure 9A).51 In another study, Wang et al. used FuGENE HD reagent (Promega) to cotransfect three plasmids expressing Cas9 and sgRNAs targeting Tet1, Tet2, and Tet3 to the mouse embryonic stem (ES) cells in the study of one-step generation of animals carrying multiple gene mutations, resulting in 20% of cells having mutations in all six alleles of the three genes (Figure 9B).54 Another group (Ousterout et al.) demonstrated the multiplex gene-editing capability of the Cas9 and sgRNA expression plasmids by electroporation to facilitate the correction of dystrophin mutations that cause DMD and could correct up to 62% of DMD mutations.179 As compared to the use of separate plasmids, construction of all of the sgRNA expression cassettes into the same vector brings a higher probability of codelivery and coexpression. For example, Sakuma et al. built an “all-in-one” system containing seven gRNA expression cassettes and a Cas9 expression cassette to achieve multiple gene editing in human cells with the efficiency ranging from 4% to 36% for each individual target (Figure 9C).180 However, this approach would increase the plasmid size and the difficulty for vector construction; the cloning effort and downstream clonal isolation can be burdensome. Alternatively, the sgRNA can be generated by in vitro transcription or chemical synthesis.10,57 sgRNAs generated by in vitro transcription or chemical synthesis should have a fast onset but may be easily degraded. Protection via complexation with a carrier or chemical modification should be helpful to minimize the degradation, which will be discussed in the following section. 5.1.2. Current Delivery of Cas9 Expression mRNA. Using Cas9 mRNA instead of DNA bypasses the requirement of nuclear entry of DNA (Figure 8), as mRNA translation takes place in the cytoplasm. Moreover, use of Cas9 mRNA results in transient expression and eventually complete removal from the body. Both are advantageous in reducing off-targeting and integration, although in principle a too short expression may also result in low efficiency. So far, the codelivery of Cas9 mRNA and sgRNA by microinjection has led to genomic cleavage in mouse SSCs,11 single-cell mouse zygotes,10,61,181 and one-cell zygotes of cynomolgus monkeys,55 resulting in biallelic mutation/correction in newborn mice (Figure 10A) or generation of gene-modified cynomolgus monkey (Figure 10B). These results showed that such physical delivery of Cas9 mRNA and sgRNA produced off-target mutations in only rare instances. However, microinjection is practical only for in vitro and certain applications. Liang et al. used the Lipofectamine-mediated transfection of Cas9 expression plasmid, Cas9 mRNA, or Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) to edit a variety of mammalian cells.22 They compared the gene editing efficiency of three types of Lipofectamine with electroporation. Interestingly, the Cas9

mRNA transfection mediated by Lipofectamine produced the highest gene-editing efficiency in most of the cell lines studied as compared to the Cas9 plasmid and protein configurations (Table 2). This work also provided the evidence that the use of Table 2. Comparison of Plasmid DNA, Cas9 mRNA/gRNA, and Cas9 RNP Transfection and Resulting Editing Efficiencies As Measured by GCDa Assay in a Variety of Cell Linesb plasmidc cell linesg HEK293FT U2OS mouse ESCs human ESCs (H9) human iPSCs N2A Jurkat K562 A549 human keratinocytes (NHEK) human cord blood cells CD34+

mRNAd

proteine

lipid

electro

lipid

electro

lipid

electro

49 15 30 0 0 66 0 0 15 0

49 50 45 8 20 75 63 45 44 30

70 21 45 20 66 66 0 0 23 0

40 24 20 50 32 80 42 27 29 50

51 18 25 0 5 66 0 0 20 0

88 70 70 64 87f 82 94f 72 66 35

n/a

0

n/a

0

n/a

24

a

GCD, GeneArt genomic cleavage detection. bReprinted with permission from ref 22. Copyright 2015 Elsevier B.V. cThe dose for plasmid transfection by Invitrogen Lipofectamine 2000 is 1 μg/mL plasmid DNA per well in a 24-well plate. The electroporation was performed with the standard dose of Neon Transfection System from Inivitrogen Corp. The indel was measured by GCD at 48 h after transfection. dThe dose for Cas9 mRNA/gRNA transfection by Invitrogen Lipofectamine 2000 is 0.5 μg of mRNA followed by addition of 50−100 ng of gRNA per well in a 24-well plate. The electroporation was performed with the standard dose of Neon Transfection System from Inivitrogen Corp.. The indel was measured by GCD at 48 h after transfection. eThe dose for Cas9 protein/gRNA transfection by Invitrogen Lipofectamine 2000 is 0.5 μg of protein followed by addition of 120 ng of gRNA per well in a 24-well plate. In electroporation study, 24 μg of purified Cas9 protein and 4.8 μg gRNA were used for 4.8 × 106 cells. The indel was measured by GCD at 48 h after transfection. fConfirmed by sequencing. gsgRNA targets are HPRT for human cell lines and Rosa 26 for mouse cell lines.

Cas9 mRNA or protein would produce lower off-target cleavage rate than the use of plasmid DNA. However, the Lipofectamine for in vitro application is not suitable for in vivo applications because of its toxicity. Yin et al. first reported the systemic delivery of Cas9 mRNA to hepatocytes by lipid-like material in conjunction with the delivery of sgRNA/HDR template by AAV.25 This treatment can efficiently cure Fahmut/mut mice (Figure 10C) with a 6% Fah correction, suggesting the therapeutic potential of systemic administration of viral and nonviral CRISPR constructs in combination. Recently, Miller and co-workers demonstrated the nonviral CRISPR/Cas gene editing in vitro and in vivo enabled by codelivery of Cas9 mRNA and sgRNA via zwitterionic amino lipid nanoparticles.26 5.1.3. Current Delivery of Cas9 Protein. Liang’s study reported that the nuclease-mediated indel rates for Cas9 RNPs based on electroporation could be up to 94% in Jurkat T cells and 87% in iPSCs for a single target (Table 2).22 When using this approach for multigene targeting in Jurkat T cells, they found that two-locus and three-locus indels were achieved in approximately 93% and 65% in the resulting isolated cell lines, 9887


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Figure 11. Genome engineering based on Cas9 RNPs delivery. (A) Schematic illustration (1) and genome editing efficiency (2) of intracellular Cas9 RNPs delivery of a method termed iTOP, for induced transduction by osmocytosis and propanebetaine, in which a combination of NaCl hypertonicity-induced macropinocytosis and a transduction compound (propanebetaine) induces the highly efficient transduction of proteins into a wide variety of primary cells. I, macropinocytotic uptake; II, intracellular release. DPH7, a gene that was identified as an essential host factor for Diphtheria toxin lethality. Biallelic deletion of DPH7 renders human cells resistant to Diphtheria toxin-induced cell death, providing a simple and effective means of identifying knockout cells and measuring the efficiency of biallelic gene knockout upon iTOP-mediated CRISPR/Cas9 delivery. Reprinted with permission from ref 186. Copyright 2015 Elsevier Inc. (B) Rational engineering of the Cas9 protein and arginine nanoparticles (ArgNPs) for intracellular delivery of the Cas9 protein or Cas9-RNP via membrane fusion. (1) Engineering Cas9 to carry an N-terminus E-tag and a C-terminus nuclear localization signal (NLS). (2) Chemical structure of ArgNPs. (3) Schematic showing nanoassembly formation by Cas9En-RNP and ArgNPs. (4) Delivery of Cas9En via a membrane fusion mechanism. Fusion of nanoassemblies to the cell membrane may facilitate direct release of the protein payload intocytoplasm, bypassing endosomes. (5) Efficient gene editing resulted from Cas9En-RNP delivery. Delivery of Cas9E15RNP to target AAVS1 and PTEN genes in HeLa cells resulted in efficient gene editing, as determined by indel (insertion and deletion) assay. Lane 1: Cas9E15-RNP/ArgNPs. Lane 2: Cas9E15-RNP. Lane 3: Cells only. Indel efficiency is given in percentage. Reprinted with permission from ref 32. Copyright 2017 American Chemical Society.

respectively. In principle, delivering Cas9 nuclease in protein form enables the swiftest editing as there is no need for transcription or translation (Figure 8). Moreover, the Cas9 protein form also obviates the requirement for codon optimization or promoter selection for optimal expression. Kim et al. directly delivered purified RNPs by electroporation into “hard-to-transfect” fibroblasts and pluripotent stem cells and induced site-specific mutations at frequencies up to 79%, which also reduced the off-target mutations as compared to plasmid transfection.182 Furthermore, they reported that RNP delivery was less stressful to human embryonic stem cells, producing at least 2-fold more colonies than plasmid transfection. T-cell genome engineering holds great promise for immunotherapies of tumor, HIV, primary immune deficiencies, and autoimmune diseases, but the genetic

manipulation of primary human T cells is a big challenge. Schumann et al. expanded the Cas9 RNP electroporation delivery to gene knock-in of mature human T cells with up to ∼20% efficiency.183 Considering the limitation of electroporation delivery, Ramakrishna et al. developed the simple cellpenetrating peptide (CPP)-conjugated recombinant Cas9 protein and CPP-complexed guide RNA for endogenous gene disruptions in human cell lines, with an indel frequency of 2.3− 36% and reduced off-target mutations relative to plasmid transfections.30 It should be noted that the size of the CPPCas9:sgRNA complex is about 400 nm, exceeding the most suitable size (i.e., a diameter of less than 200 nm) for in vivo delivery to tumors or organs such as liver.184,185 D’Astolfo et al. reported a CPP-independent protein intracellular transduction method termed iTOP for delivery of recombinant Cas9 RNPs 9888


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Figure 12. Nonviral Cas9 delivery. (A) Cationic lipid-mediated delivery of Cas9 proteins enables efficient genome editing in vitro and in vivo. (1) Schematic illustration of the strategy for delivering proteins into mammalian cells by fusion or noncovalent complexation with polyanionic macromolecules and complexation with cationic lipids. (2) EGFP gene knockout efficiency for delivery of Cas9 RNPs to U2OS EGFP reporter cells. (+36)dGFP-NLS-Cas9 and (−30)dGFP-NLS-Cas9 are the fusion Cas9 proteins of either highly cationic GFP variant protein (+36)dGFP or highly anionic GFP variant protein (−30)GFP to Cas9. NLS, nuclear localization signal. (3) EGFP gene knockout efficiency for in vivo delivery of RNPs. Yellow boxes in the lower panels highlight hair cells that have lost GFP expression. All scale bars (white), 10 μm. Reprinted with permission from ref 23. Copyright 2014 Nature Publishing Group. (B) Schematic illustration of brain tumor modeling by somatic CRISPR/Cas9-mediated tumor suppressor disruption. (1) Somatic deletion of the Ptch1 or multiple genes (Trp53, Pten, Nf1) locus induces medulloblastoma (MB, mediated by Cas9 and sgRNA expression plasmid delivery based on PEI in vivo transfection or electroporation. (2) H&E staining of a cerebellar tumor induced by transfection of a Trp53−/− animal with gPtch1.1/Cas9 plasmid delivery (right). Sagittal section of a WT mouse cerebellum after in utero electroporation of gPtch1.1/Cas9 showing a large tumor (left). Scale bars, 200 μm. Reprinted with permission from ref 150. Copyright 2015 Nature Publishing Group.

into primary cells,186 depending on a combination of NaCl hypertonicity-induced macropinocytosis and a transduction compound (propanebetaine) (Figure 11A). They demonstrated that iTOP could mediate the delivery of recombinant Cas9 RNPs with 70% gene knockout efficiency in a nonintegrative manner (Figure 11A). Self-assembled DNA nanoclews were specially designed as another CPP independent carrier for the efficient delivery of Cas9 RNPs in vitro and in vivo.34 The DNA nanoclews loaded with Cas9 RNPs showed a gene knockout efficiency of approximately 36%, as compared to only 5% for PEI. Another study described the engineering of Cas9 protein with a negatively charged E-tag to facilitate complexation with arginine-decorated Au nanoparticles. The assembly of these nanoparticles into submicrometer particles led to direct cytosolic delivery and achieving a high gene editing efficiency up to 30% in vitro (Figure 11B).32 All of these proof-ofprinciple studies highlight the advantage of the Cas9 protein form for the therapeutic gene correction or gene replacement if efficient delivery systems are available. However, the afford-

ability and purity of Cas9 protein are somewhat hindering the development of effective Cas9 protein delivery systems. In addition, in vivo delivery of the protein form may induce nonspecific nuclease action-mediated toxicity or trigger humoral immunity to Cas9.173,174 5.2. Viral versus Nonviral Delivery

Viral vectors can be widely used in gene transfer due to the relatively high efficiency and potentially sustained effect through the integration into the host genome. LVs have been used to constitutively express Cas9 and/or gRNAs in human and murine cells.17,187,188 In one study, LV vectors delivered 64 751 unique sgRNAs in a genome-scale CRISPR/Cas9-based knockout library for enabling screening of gene function in human cancer cells and PSCs.187 However, reports of LVs causing viral DNA integration into genome and resulting unwanted genetic mutations pose concerns about their safety in clinical applications.189 In conventional gene therapy, the integrase-dependent mechanism of LVs is a crucial feature for 9889


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nanoclews34 has demonstrated the feasibility of Cas9/gRNAs delivery. Lipofectamine-mediated transfection for CRISPR/ Cas9 in vitro genome editing is commonly adopted for its high transfection efficacy. As we mentioned above, Lipofectamine performed well in the Cas9 expression plasmid, Cas9 mRNA, and Cas9 protein delivery.22 In Zuris’s study, delivery of Cas9 RNPs resulted in an 80% genome modification in vitro with a substantially higher specificity as compared to DNA transfection (Figure 12A).23 This approach also mediated efficient delivery of Cas9 RNPs into the mouse inner ear in vivo, achieving 20% Cas9-mediated genome modification in hair cells (Figure 12B). Zuckermann et al. established brain tumor modeling by somatic CRISPR/Cas9-mediated tumor suppressor disruption (Figure 12B).150 They used in vivo PEI transfection or electroporation for Cas9/sgRNA expression plasmids to delete single (Ptch1) or multiple genes (Trp53, Pten, Nf1) in the mouse brain, resulting in the development of medulloblastoma and glioblastoma, respectively. Although many nonviral vectors have shown good performance on the Cas9 and sgRNA delivery, most of them can only be used in the in vitro, ex vivo, or local in vivo administration. Nonviral delivery of Cas9 and sgRNA based on systemic administration remains unsatisfactory. It is probable that new carriers have to be custom-designed to suit the characteristics of the Cas9/ sgRNA complex, but a lot can be accomplished by adapting existing systems that have been extensively optimized for plasmid DNA and siRNA delivery.

achieving stable expression of transferred genes to maintain the complementation of genetic defects in dividing cells. However, when LVs were used for CRISPR/Cas9 delivery, the integration capability of LVs should be disabled as sustained presence of Cas9s and sgRNAs may cause higher off-target effects.190 AAV, a nonpathogenic parvovirus, is proved to be efficient in gene delivery because of their low immunogenicity, low propensity of integration into the host genome, and well-characterized specificity.191 In contrast to LVs, AAVs lack integration machinery. When they enter the cell nuclei, most of the viral genomic DNA are in an episomal state. As a result, the nonintegrated DNA would be diluted in actively dividing cells during mitosis, leading to a transient gene expression. However, in predominantly postmitotic cells (e.g., neuron, muscle fiber, and hepatocyte), the genomic DNA delivered by AAVs can be maintained stably in a nonintegrated form to mediate persistent gene expression.192 AAVs typically have a low frequency of integration at around 0.1−0.5%, but they have shown integrated frequencies as high as 5−10% in some tissues, such as mouse liver.193 Recently, AAVs technology has been deployed for delivering CRISPR/Cas9 components to investigate gene function in the mammalian brain and to model lung cancer development.31,193 However, it is difficult to transduce SpCas9 and multi gRNAs together with AAV vectors due to a restricted packaging capacity of 4.7 kbps.194 As shorter Cas9 variants can resolve this problem, several of them (e.g., CRISPR from Staphylococcus aureus, Streptococcus thermophiles, and Neisseria meningitidis) have been applied to human cells.20,51,195 However, shorter Cas9 variants require longer and more complicated PAM sequences, restricting DNA target sites of choice. Alternatively, Cas9 and sgRNA can be packaged in two separate viral vectors to overcome the packaging size restriction of AAV vectors,196 but separate vectors are not convenient when series of sgRNAs are needed at the same time in multiplex gene-editing. Another strategy to solve the packaging restriction of AAV vectors is to establish a splitCas9 platform by packing the Cas9 C-terminal (Cas9-C) expression cassette and Cas9 N-terminal (Cas9-N) expression cassette into two separate AAV vectors, and reconstituting the expressed Cas9-C and Cas9-N into functional Cas9 in cells to perform genome editing with efficiency comparable to that of wild-type Cas9.173,197 Nonviral approaches for introducing CRISPR/Cas9 show the potential to address many of the aforementioned limitations with viral vectors.21 Unlike viral vectors, nonviral vectors have lower immunogenicity.198,199Additionally, they are not limited by the packaging restrictions, are easier to synthesize, and are compatible with the simultaneous delivery of multiple sgRNAs. Through carrier design alteration and targeting ligand decoration, nonviral vectors can deliver therapeutic nucleic acids to target tissues or cells. Recent nonviral approaches to introduce Cas9/sgRNAs into cells in vitro and in vivo have presented a glimpse of the potential both chemical and physical methods (Figure 1 and Table 1). For physical methods, microinjection, electroporation, and cell mechanical deformation have demonstrated the potency of Cas9/sgRNAs delivery. For example, Han et al. reported a microfluidic membrane deformation method to deliver sgRNA and Cas9 into different cell types and achieved >90% gene knockout efficiency.178 For chemical methods, the use of lipid-based nanoparticles,22−26 polyethylenimine (PEI),27 Ca3(PO4)2,28 FuGENE 6 transfection reagents,29 cell-penetrating peptides (CPP),30 7C1 nanoparticles,31 cationic arginine gold nanoparticles,32 “core-shell” artificial virus,33 and DNA

6. POTENTIAL STRATEGIES TO ADAPT CURRENT NONVIRAL DELIVERY SYSTEMS FOR CRISPR/Cas9 DELIVERY 6.1. Potential Physical Approaches for CRISPR/Cas9 Delivery

As we have shown above, both physical and chemical approaches can be used to deliver Cas9 expression plasmid, mRNA, or protein, as well as sgRNAs for in vitro or in vivo genome editing (Figures 1 and 8). Herein, we first overview the most commonly used physical methods for DNA/mRNA/ protein transfer and assess the potential advantages and disadvantages of such approaches as compared to other chemical systems. The physical methods involving microinjection, electroporation, hydrodynamic injection, or other strategies such as membrane deformation and sonoporation rely on delivering the DNA/mRNA/protein into the cytoplasm/nucleus via transient “opening” of the cell membrane. 6.1.1. Microinjection. The most direct method to introduce DNA/mRNA/protein into cells is microinjection. It bypasses the extracellular and cytoplasmic barriers to deposit the molecular cargoes in a controlled manner into the nucleus/ cytoplasm. Unsurprisingly, microinjection shows high efficiency, sometimes up to 100%.200 Moreover, the total quantity of injected Cas9 elements/sgRNAs is known, thereby more easily controlling and reducing the off-target effect. In addition, this approach is not limited by the molecular weight and size of Cas9 elements/sgRNAs, which is helpful for the large-size Cas9 expression plasmid/mRNA and the full Cas9 protein with a 130 kDa molecular weight. So far, this technique has been efficiently used to edit genes in various cells or animals (including zebrafish,201 rat,153 mouse,202 rabbit,203 sheep,204 and monkey55). However, microinjection is a laborious technique that can only handle a few hundred cells per experiment. Therefore, 9890


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possesses primary, secondary, and tertiary amines, and are therefore endowed with a high charge density and more importantly pH-buffering capacity. The former facilitates condensation of the pDNA to a small size, and the latter promotes escape of the polymer-DNA nanoparticles (polyplexes) from the endolysosomal compartment due to the proton sponge effect. The major problem with 25 kDa branched PEI transfection is excessive cytotoxicity. The cytotoxicity can be reduced by using linear PEI and lower molecular weight,212,213 or by chemical modification with moieties such as PEG. Several PEI-based vectors are under clinical evaluation for human cancer therapy.214−216 These studies will help establish PEI-based carriers for Cas9 gene delivery.27,30,150 Chitosan is another polymeric candidate for Cas9 gene delivery. As a natural biopolymer, the polysaccharide enjoys low toxicity.217 The most critical factor to influence the gene transfer efficiency of chitosan is the molecular weight. Our study showed that high molecular weight chitosan formed more stable polyplexes due to a chain entanglement effect.218 Other groups also reported that increasing the molecular weight of chitosan could improve the gene transfection efficiency.219,220 However, for Cas9 plasmid transfection, chitosan with too high a molecular weight (>200 kDa) may condense the large Cas9 plasmid too tightly and hinder their release. In addition to molecular weight, the deacetylation degree of chitosan also plays a significant role in affecting the transfection efficiency of chitosan.218 Huang et al. reported that chitosan with higher molecular weight (Mw 213 kDa) and deacetylation (88%) showed the highest transfection efficiency in vitro for various cell lines, as compared to the chitosan with lower molecular weight (Mw 10−98 kDa) and lower deacetylation (46% and 61%).219 However, our data showed that chitosan with a moderate degree of deacetylation (70% and 62%) was much more efficient for in vivo gene transfection than chitosan with a higher degree of deacetylation (90%).218 These studies highlight the importance of finding the optimal balance of DNA condensation to form small and stable polyplexes versus DNA release to achieve effective gene delivery. Nevertheless, chitosan may not be efficient for Cas9 plasmid delivery because of its low transfection efficiency for large genes. Xu et al. reported low transfection efficiency against chondrocytes in the delivery of a relatively large plasmid (8−10 kb) encoding osteogenic protein (OP)-1, although the same chitosan carrier has proved efficient for the EGFP plasmid.221 Oliveira et al. improved the transfection for such large plasmid through combination with phiC31 integrase,222 but incorporating the Cas9 gene into the genome would be undesirable. Hence, in the future, enhancing the dissociation of the chitosan/Cas9 polyplex instead of incorporating the gene to the genome with the help of nonspecific integrase would be preferable. PLL is a homopolypeptide of the basic amino acid lysine. Nanomedicines based on PEG-b-PLL carrier have been applied to the treatment of cystic fibrosis in clinical trial although without success.223 Gene carriers based on PEG-b-PLL show equivalent ability for delivery of plasmids ranging in size between 5.3 and 20.2 kb,224 indicating that nonviral vector based on PLL may be a good choice for Cas9 delivery. Lipid-based carriers composed of cationic lipids like N-[1(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 1,2-di-

while microinjection is valuable for single cell genome editing, it is impractical for most applications where millions or even billions of cells have to be edited. 6.1.2. Electroporation. Electroporation is another popular physical method to transfer nucleic acid into cells. It achieves intracellular delivery via transient disruption of the lipid bilayer constituting the plasma membrane. Feasible for intracellular delivery of even small nanoparticles, electroporation is applicable to hydrodynamic volumes of tens of nanometers, and hence suitable for Cas9 delivery. Up to now, electroporation has performed well in CRISPR/Cas9 delivery in vitro and in vivo.11,13,150 It will likely become one of the most commonly used techniques for CRISPR/Cas9 delivery in the future due to its efficiency and safety. However, cell death and loss of cell stemness can sometimes be problematic, but optimization of the electroporation parameters and the composition of the electroporation medium may minimize the issue. 6.1.3. Hydrodynamic Injection. Hydrodynamic injection is a powerful nonviral method for in vivo gene delivery in rodents.205 It delivers DNA plasmids into cells by a high pressure in a short time to form transient pores in the cell membrane. With hydrodynamic injection into the tail vein, efficient transfection in liver, kidney, lung, and heart can be achieved. Recently, this technique has been applied to mutate cancer genes in the mouse liver,147 correct a hereditary tyrosinemia mutation and phenotype,57 or disrupt the HBV in vivo.27,206 However, hydrodynamic injection requires a large injection volume and causes severe damage to the liver. Up to now, there has been only one human clinical trial using hydrodynamic injection. It is clear that this procedure needs a significant improvement before further evaluation for translation. Other vector-free physical delivery systems such as sonoporation,207−209 laser irradiation,210 or membrane deformation175,211 can also be used for CRISPR/Cas9 delivery. They work under a mechanism similar to that of other physical delivery methods by creating transient holes in the cell membrane, and likewise face issues of potential cell damage. Moreover, physical methods usually rely on naked DNA/ mRNA/protein without any carrier to protect the cargos. When administered directly into tissues or systemic circulation, the naked cargo is also vulnerable to enzymatic degradation and rapid clearance. Therefore, the alternatives of chemical nonviral delivery are likely to play a predominant role in the near future. 6.2. Potential Chemical Approaches for CRISPR/Cas9 Delivery

6.2.1. Potential Vectors for Cas9 Expression Plasmid Delivery. Theoretically, delivery of the Cas9 plasmid using nonviral vectors should follow the same principles as for other plasmids. The nanocomplexes would be formulated by electrostatic interactions, optimized for small size, maximal cellular uptake, protection from degradation, and opportune intracellular unpacking. However, Cas9 expression plasmids are large plasmids. Parameters such as charge ratio (N/P), particle zeta potential, particle size, DNA release rate, and endosome escape may have to be optimized differently. We review the current vectors to predict their potential for CRISPR/Cas9 delivery. Polymeric carriers or lipid-based carriers dominate the field. Polymeric carriers enjoy chemical diversity and versatility. The most common examples of polymeric DNA vectors are PEI, chitosan, and poly(L-lysine) (PLL). Branched PEI 9891


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conventional pDNA carriers would be sufficient. It is difficult to study the intracellular unpacking and degradation of polyplexes and lipoplexes. A number of commercially available lipoplexes, such as Lipofectamine, have been used in vitro for transfection of mRNA,22,232,233 although these reagents have limited utility in vivo due in part to their toxicity and poor transfection potency. Proprietary cationic lipids MegaFectin and TransIT reportedly can deliver mRNA in vivo effectively.232,234 Other lipids or lipid-like materials showing efficiency including the cationic DOTAP and the zwitterionic phospholipid DOPE formulation,235,236 DLinDMA/DSPC/cholesterol/PEG formulation,237 and C12-200 formulation.238 Polymeric carriers, such as chitosan,239 Protamine,240,241 PEI,236 poly(2-dimethylaminoethyl methacrylate) (PDMAEMA),242 and poly(β-amino esters) (PBAEs),232 have been adapted from siRNA/DNA delivery to mRNA delivery in recent years. Mahiny et al. used chitosan-coated poly(lactic-co-glycolic acid) (PLGA) for ZFN mRNA delivery to realize site-specific genome editing in lung,239 which may also work for Cas9 mRNA delivery. A linear PEI derivative, jetPEI, commonly used for DNA and siRNA transfection can also be used for mRNA transfection, although showing a lower efficiency than lipoplexes.236 Previously, we showed that mRNA delivered by Stemfect transfection reagent performed better than naked mRNA when administered intranasally and intravenously.243 Much of the current research concerning in vivo delivery of mRNA targets immunization. One example is the ongoing clinical trial using protamine/mRNA complexes for intradermal injection to treat metastatic melanoma.244 Another example is intranasal mRNA delivery by mixing the mRNA with PBAEs to form a core and then coating it with a positively charged lipid bilayer shell.232 Single-strand mRNAs can serve as ligands for the pattern recognition toll-like receptors TLR7 and TLR8 to activate these pathways, which is a valuable characteristic for mRNA vaccination, but undesirable for other Cas9 mRNA applications. Therefore, the development of better delivery systems for Cas9 mRNA remains an unmet need. 6.2.3. Potential Vectors for Cas9 Protein Delivery. Several studies have compared the gene editing efficiency of different Cas9 formats: pDNA, mRNA, or protein.22,182,245 Cas9 RNP delivery, which skips the expression of protein and sgRNA in cells (Figure 8) and avoids the possibility of undesired DNA integration into the genome, exhibited a significantly lower off-target cleavage rate.22,30,182 Although direct delivery of Cas9 protein into the cells would facilitate the CRISPR/Cas9 genome editing, efficient intracellular protein delivery remains a challenge because of its in vitro and in vivo instability, immunogenicity, and short half-life.16,246 In addition, the large size and the low efficiency of endosomal escape of protein would also hinder its efficient delivery.247 Successful intracellular delivery and subsequent nuclear translocation of both sgRNA and Cas9 protein into cells are indispensable for efficient genome editing.182 Up to now, most of the gene-editing systems based on Cas9 protein are delivered in the form of Cas9 RNP, which can be readily prepared by incubating Cas9 protein with either sgRNA or dual RNA consisting of crRNA and tracrRNA.22,34,182,183,248−250 Only a few studies have reported the separate delivery of Cas9 protein and sgRNA in two steps.30 Delivery of preassembled Cas9 RNP can achieve a better editing efficiency and reduced frequency of off-target effect by immediately cleaving the chromosomal target sites after transfection and undergoing rapid degradation by endogenous proteases thereafter.249 Nonetheless, it remains

myristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), and DC-cholesterol are widely used as nonviral gene carriers for their high transfection efficiency.225 The lipid-DNA complexes (lipoplexes) usually have small and relatively uniform size, and are capable of binding or encapsulation DNA efficiently to transfect cells. The poor colloidal stability, rapid clearance, and cytotoxicity induced by the cationic lipid can be solved by incorporation of neutral lipids. As we mentioned above, lipoplexes have shown good performance on the Cas9 expression plasmid/mRNA/protein and sgRNA delivery, but most of them can only be used in vitro, ex vivo, or local in vivo administration.22−24,51,52 We speculate that their potential for systemic delivery of Cas9 will be pursued intensively in the near future because of their immense popularity in the gene therapy arena.199 Up to now, there is still no report about using inorganic carriers for Cas9 expression plasmids delivery. However, inorganic nanoparticles such as silica nanoparticles,226 gold nanoparticles,227 or carbon nanotube228 have shown good performance on many gene delivery applications, which can be attractive candidates for Cas9 plasmid delivery for several reasons. As compared to polyplexes and lipoplexes, inorganic nanoparticles are more colloidally stable and easier to control with respect to composition, size, and size distribution. What is more, the physicochemical properties of some inorganic nanoparticles have been shown to be unaffected by freezedrying in the presence of various cryoprotectants, offering an appealing method for storage and addressing a critical issue for eventual translation. Like all other delivery applications, no one carrier will overcome all of the physiological barriers and enable efficient Cas9 delivery (Figure 8). Some efforts should be put on the development of multifunctional vectors sensitive to both extracellular and intracellular microenvironment.229 For example, the multifunctional envelope-type nanodevice (MEND) developed by Harashima’s group contains a polycation/pDNA complex core and a lipid coating envelope, which can be equipped with several multifunctional modules, such as a PEG layer for longer circulation time, a ligand for active targeting, a cell penetrating peptide to increase intracellular delivery, a lipid to enhance endosomal escape, and a nuclear localization signal (NLS) for nuclear delivery.230,231 Such intelligent nanocarriers can overcome multiple barriers to deliver the therapeutics in a temporally and spatially regulated fashion. 6.2.2. Potential Vectors for Cas9 mRNA Delivery. The development of mRNA delivery systems has not been as advanced as that for DNA. A primary reason is that mRNA is more labile and vulnerable to contamination. Moreover, mRNA expression is less sustained than DNA-driven expression, which may be undesirable in certain gene therapies. This is nevertheless compensated for by the significant advantage of not having to deliver the mRNA into the nucleus, which is the major transport barrier for pDNA delivery (Figure 8). Consequently, it offers a more precise control of the dosage and may help decrease the off-target effect and minimize the nuclease-induced toxicity. Carriers for mRNA delivery must protect the mRNA against RNase-mediated degradation in the extracellular space. In this respect, the conventional carriers intended for pDNA delivery should suffice for this purpose. Once endocytosed, the need to minimize degradation in the endolysosomal compartment before unpacking in the cytoplasm should be even greater than that for pDNA because of the mRNA fragility. In that respect, it is uncertain if the 9892


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Figure 13. Nonviral protein delivery. (A) Three general loading strategies for protein delivery. (B) Various types of nanovehicles used for intracellular protein delivery. Reprinted with permission from ref 247. Copyright 2011 Royal Society of Chemistry.

gold nanorod), and protein-mediated nanocarriers (Figure 13B).247 We speculate that existing platforms for protein delivery will hold great potential for both Cas9 protein and Cas9 RNP delivery. However, the opposite surface charge of Cas9 protein (positive) and Cas9 RNP (negative) should be taken into consideration in designing the delivery systems.34 Because of the importance of efficient intracellular delivery of Cas9 to gene editing, cell membrane-penetrating and nucleartargeted Cas9 delivery is favorable as it facilitates direct transport of the endonuclease into the nucleus. An alternative approach is to use NLS-attached Cas9 protein, which can mediate the transport of Cas9 or Cas9 RNP into the nucleus. For in vivo delivery, the stability of Cas9 in blood versus its intracellular release should also be taken into consideration. Stimuli-responsive designs for precise “on demand” release of Cas9 would be an attractive approach. We envision that continual improvement of Cas9 and Cas9 RNP delivery based on nonviral vectors will make it an efficient genome-editing system for both fundamental studies and clinical gene therapy. 6.2.4. Combination of Viral Delivery for Cas9 Expressing Cassettes and Nonviral Delivery for sgRNAs. The effort to include multiple sgRNA-encoding sequences in conjunction with that of Cas9 into a viral vector is not trivial. Because there exist many efficient nonviral vectors for siRNA and miRNA delivery, some of which are already under clinical trial,254,255 it would be logical to combine viral and nonviral delivery for Cas9 and sgRNA respectively. Recently, Platt et al. tested the feasibility of gene editing with nonviral sgRNA delivery by tail-vein injection of endothelial-targeting 7C1 nanoparticle with two sgRNAs targeting the endothelial-specific

unclear as to which mode of delivering the Cas9 protein and sgRNA, separate or complexed, would yield the best result. Further investigations should be performed to determine these two methods so that the efficiency of Cas9 protein editing can be rationally optimized. Generally, there are three main loading methods for protein delivery: direct conjugation of protein with carriers, physical adsorption, and emulsion-based encapsulation (Figure 13A).247 PEGylation of proteins has been widely explored for evading the reticuloendothelial system (RES), increasing the systemic circulation time, and preventing degradation by proteolytic enzymes.251 There are various FDA-approved PEG-protein conjugate drugs.252 Genetic fusion of the protein with a transduction domain and chemical conjugation of cellpenetrating peptides are the most commonly used approaches to increase cellular uptake.30 However, covalent conjugation of protein is nonspecific in most cases, thereby leading to deleterious effects on protein activity. Another commonly used method is spontaneous assembly based on electrostatic or van der Waals interactions between the protein and the carrier.247 Moreover, water/oil/water (w/o/w) double emulsion-based encapsulation has also been studied for protein delivery.253 Serving as a shield to protect proteins against premature degradation and biological environment-induced denaturation, frequently used nanocarriers for intracellular protein transduction include lipid-containing colloidal nanoparticles (e.g., liposomes and solid lipid nanoparticles), polymeric nanomaterials (e.g., polymersome, nanogel, and micelle), inorganic nanovehicles (e.g., carbon nanotube, quantum dot, mesoporous silica, magnetic nanoparticle, and 9893


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Figure 14. (A) Sequence of the IL2RG sgRNA loaded into Cas9 and bound to its DNA target site. Red flags represent the chemically modified nucleotides. (B) Chemical modifications of sgRNAs. (C and D) Gene disruption by mutagenic NHEJ as measured by deep sequencing of PCR amplicons (C) or gene addition by HDR at the three loci IL2RG, HBB, and CCR5 in K562 cells induced by Cas9 in combination with synthetic sgRNAs (D). Light shade, 1 μg sgRNAs; dark shade, 20 μg sgRNAs; gray bars, 2 μg of plasmid encoding both sgRNA and Cas9 protein. (E) Gene disruption at the CCR5 target in stimulated primary human T cells. (F) Gene disruption at the IL2RG and HBB targets in CD34+ HSPCs. (G) Indel frequencies of one or two CCR5 sgRNAs in T cells and CD34+ HSPCs. Reprinted with permission from ref 245. Copyright 2015 Nature Publishing Group.

applications, the first extracellular barrier is various nucleases/ proteases existing in blood. The in vivo half-life of naked protein ranges between minutes and hours,256 whereas naked pDNA degrades in minutes and small RNA in seconds in systemic circulation. As we mentioned above, the resistance to nuclease-mediated degradation of sgRNAs/Cas9 mRNAs might be enhanced by chemical modifications.245,257 However, naked modified-RNAs still have to face rapid clearance by the renal system. Potential recognition by the host immune system is another challenge. Cationic polymers or lipids used to condense negatively charged Cas9 plasmid/mRNA/protein are also easily recognizable by the RES.258,259 Encapsulating the Cas9 elements in a nanoparticle with the PEG/zwitterionic materials or “self” peptides modification is desirable, which can protect Cas9 elements from degradation, improve circulation time, prevent recognition as a foreign invader, and help escape host immune clearance.260−262 The barrier of efficient accumulation of Cas9 elements/particles in the target tissue is the next formidable challenge. We assume that the previously applied strategies for enhancing particle accumulation in the

gene intercellular adhesion molecule 2 (Icam2) to constitutive Cas9-expressing mice with a dose of 1.5 mg/kg sgRNA and total of five injections.31 This led to indel formation at the target sites of Icam2 and reduced levels of Icam2 protein expression in pulmonary (60%) and cardiovascular (70%) endothelial cells in mice. Although the combination of viral and nonviral delivery for Cas9 and sgRNA may show some advantages such as higher flexibility, concerns for potential host immune response caused by Cas9 expression may still persist and remain to be determined.

7. CRITICAL BARRIERS TO NONVIRAL DELIVERY OF CRISPR/Cas9 7.1. Extracellular and Intracellular Barriers of Nonviral Delivery of CRISPR/Cas9

Following administration, Cas9 elements delivered by nonviral vectors should remain stable in, and transport through, extracellular barriers before potentially being taken up by cells, and in cases for Cas9 expression plasmids, must traffic through the cell to the nucleus (Figure 8). In systemic 9894


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Figure 15. (A) Chemical structures of unmodified and modified nucleotides. (B) Step-wise synthesis of 32- and 29-mer scrRNA. (C) Full sequences of VEGF-A-targeting truncated scrRNAs and their relevant gene disruption efficiencies. Reprinted with permission from ref 257. Copyright 2015 National Academy of Sciences of the United States of America.

turn contribute to its application in many therapeutic platforms.271,272 In general, frequently utilized chemical modifications fall into three categories: (1) internucleotide linkage modification, (2) sugar modification, and (3) nucleobase modification.270,273 Chemical modification is a robust and feasible method for manipulating and optimizing the properties of sgRNA. The rationale and strategies for chemical modification of RNAs are almost the same. So far, comprehensive studies have been carried out on the effect of chemically synthesized and modified siRNA, 270,274−276 mRNA,232,239 miRNA,277,278 anti-miRNA RNA oligonucleotide,279−281 and RNA aptamer,282,283 whereas only a few studies have focused on improving the efficiency of CRISPR/Cas9 systems by varying the chemical structures of sgRNA nucleotides. Hendel et al. chemically synthesized 100-mer sgRNAs with chemical modifications at the 5′ and 3′ terminal positions (Figure 14A), and evaluated their effects on genome editing efficacy in human primary T cells as well as CD34 + hematopoietic stem and progenitor cells (HSPCs).245 Three chemical modifications including 2′-O-methyl (M), 2′-Omethyl 3′ phosphorothioate (MS), and 2′-O-methyl 3′ thioPACE (MSP) (Figure 14B) were tested because of their reported nuclease stability and immunostimulatory properties.270,284 As compared to unmodified sgRNA, the chemically modified ones retained high specificity and demonstrated improved gene editing efficiency when codelivered with Cas9encoding plasmid/mRNA or Cas9 protein. Specifically, when delivered with the plasmid encoding Cas9 into K562 cells via electroporation, unmodified, M-modified, MS-modified, and MSP-modified sgRNAs displayed indel frequencies (for the IL2RG target) of 2.4%, 13.5%, 68.0%, and 75.7%, respectively (Figure 14C). Further dosage increase of sgRNAs by 20-fold resulted in enhanced efficiencies of MS- and MSP-modified sgRNAs to 75.3% and 83.3%, respectively. Similar results were obtained for indel frequencies at the HBB and CCR5 targets (Figure 14C), and HDR frequencies at all of the above three targets (Figure 14D). For stimulated human primary T cells, codelivery of Cas9 mRNA with either MS- or MSP-modified sgRNA achieved significantly higher efficiency than the combination of Cas9 mRNA and M-modified sgRNA, showing 48.7% and 47.9% indel frequencies, respectively (Figure 14E).

targeting tissue by negative targeting such as the enhanced permeability and retention (EPR) effect or active targeting with ligands would also be applicable to CRISPR/Cas9 delivery. Proceeding from the tissue to the cell level, vectors face the clearance/blockage of epithelial cell, endothelial cell, and the negatively charged extracellular matrix. They have to extravasate from the blood vessel and may have to penetrate deep into the tissue to meet the targeted cells. To achieve high vascular permeability and tissue penetration, nanoparticles in small size are preferred.263,264 As nucleic acids are large, hydrophilic, anionic molecules, they cannot readily traverse the hydrophobic lipid cell membrane.265 Cellular uptake is the next big challenge. After a successful internalization into the targeted cells, vectors should carry its payload (Cas9 expression plasmid/mRNA/protein and sgRNA) out of endosome. Once released from endosomal compartments, vectors with payload, except for Cas9 mRNA, must move through the cytoplasm to the nucleus. However, the cytoplasm is concentrated with proteins, microtubules, and other organelles, all of which can hinder the vector movement. Researchers have showed that pDNA directly injected into the cell nuclei expresses much better than mRNA directly injected into the cytoplasm.266 Hence, the nuclear membrane is regarded the critical barrier. NLS is often used to assist nucleic acid or protein in entering through nuclear pore complexes by active transport.267 Moreover, vector unpacking is also a potential barrier for Cas9 delivery. For polyplexes, mechanistic studies have shown that the slow vector unpacking is a likely factor for low transfection efficiency.268 7.2. Chemical Modifications To Overcome the Instability of sgRNA

As the positioning system, sgRNA plays a central role in Cas9mediated site-specific gene editing. RNA suffers from rapid degradation due to its combination of thermodynamically unstable bond and carbohydrate building block, especially in the serum and tissues with a variety of ribonucleases.257,269 Specific chemical modification of nucleotide and its analogues provides an alternative method to manipulate some pivotal properties of nucleic acid, including nuclease stability, target binding affinity, structural preference, and immunostimulatory activity.270 These chemically modified properties of RNA in 9895


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A similar trend was observed in peripheral blood CD34+ HSPCs for IL2RG and HBB targets (Figure 14F). Further, in both T cells and CD34+ HSPCs, a combination of two chemically synthesized MS- and MSP-modified CCR5 sgRNAs (termed “D” and “Q”) increased the frequencies of allele disruption (Figure 14G). Cleveland and co-workers have recently reported a series of chemically modified synthetic CRISPR RNAs (scrRNAs) for genome editing in human cells.257 These modifications include phosphorothioate (PS) backbone linkage as well as 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me), and S-constrained ethyl (cEt) nucleobase substitutions (Figure 15A). The PS modification makes the scrRNAs more resistant against nucleolytic degradation.281,285 2′-F, 2′-O-Me, and cEt substitutions are used to improve the biostability285,286 and binding affinity of scrRNA to tracrRNA.257 The 42-nucleotide crRNA with 2′-OMe and 2′-O-Me plus PS modifications showed improved gene disruption activities as compared to unmodified crRNA, yielding 4- and 7-fold increases, respectively. Nevertheless, the efficiencies were still lower than that of transcriptionproduced sgRNA (33% and 48% of the sgRNA, respectively). A combination of 2′-F and 2′-cEt modifications demonstrated the highest gene-editing activity (75% of that achieved by sgRNA). Keeping a similar chemical substitution pattern and removing 10 nucleotides from the 3′-end of scrRNA resulted in ∼42% activity of sgRNA (Figure 15B and C). A 29-mer scrRNA generated by further shortening the scrRNA from the 5′-end while skipping a modification in the seed sequence demonstrated an equal (107%) activity of sgRNA (Figure 15B and C). As a strategy combining genetic and synthetic methods for CRISPR/Cas gene-editing, this approach increases controllability over the activity of a CRISPR drug.287 One can activate or inhibit the pharmaceutical activity by introducing a short scrRNA that guides the Cas9 endonuclease to a DNA target, or a complementary oligonucleotide to the scrRNA or tracrRNA, respectively.257 Generally speaking, the chemically synthesized and modified sgRNA endows the following advantages as compared to the transcribed one: (1) enhanced stability, selectivity, and editing efficiency, (2) scalable production for potential therapeutic applications, and (3) regulatory flexibility for the design. On the other hand, the higher cost is a discouraging factor.

bulk mixing is ill suited to produce uniform nanoparticles with such fast reaction kinetics. The composition of the nanoparticles depends on how the polyelectrolytes encounter one another, which is governed by the chaotic mixing in the Eppendorf tube. Even a slight deviation of the mixing protocol is known to produce nanoparticles of differing qualities. For instance, whether adding polycation solution to the Eppendorf tube or the reverse of adding DNA solution to the polycation would make a difference; the angle one presses the tube to the Vortex Mixer also makes a difference, and so does the pressure applied. Therefore, bulk mixing introduces great variability to the quality of the nanoparticles because of the metastable preparation and nonequilibrium composition. This issue is even more acute in Cas9/sgRNA nanoparticle formation because Cas9 expression plasmid is a large plasmid and several sgRNAs are often involved. The heterogeneity in size and composition resulting from bulk synthesis would hinder the development of nonviral Cas9/sgRNA delivery in several ways: (1) It obscures mechanistic understanding of the delivery process if only a subpopulation of the nanoparticles is responsible for the phenomenon; (2) it compounds the challenge of establishing an accurate structure−function relationship; and (3) it will eventually hinder the translation of any nanodelivery system if it cannot be manufactured in a reproducible and scalable manner. One approach to better control the charge neutralization process is to do it in picoliter-sized droplets generated by a microfluidic setup.291,292 The confined diffusion in a small volume would facilitate the charge neutralization between the oppositely charged polyelectrolytes to reach equilibrium, without the influence of tumultuous and uncontrolled bulk mixing. This effective reaction may also exhaust the free polyelectrolytes within the volume, leaving a minimum of unreacted reagents, particularly the polycations that typically generate a corona on the surface of the nanoparticles. Consistent with this hypothesis, polyplexes self-assembled in picoliter-sized droplets are smaller, narrower in size distribution, more compact, lower in zeta potential, and more stable colloidally as compared to their bulk-mixing counterparts. Transfection efficiency is also higher. An important significant finding is that the amount of excess, free-floating polycation is much reduced, leading to lower cytotoxicity.291 However, the scale-up of such a microfluidic process has not been addressed properly. In principle, scale-up can be done with optimization of reactant concentration, flow rate, and droplet volume, coupled with microfluidics array designs. Another approach that might have an easier path of scale-up is 3D hydrodynamic focusing in a continuous flow.293 In this technique, a curved channel in the microfluidic device provides a centrifugal force to squeeze the third dimension of the polycation/nucleic acid interface, thereby reducing the sample volume and minimizing flocculation after the nanoparticle formation. However, this technique might not be suitable for nanoparticle formation involving the Cas9 protein as the high flow rate and shear might denature the large Cas9 protein. There are other fabrication techniques, such as flash nanoprecipitation and printing technology, that might improve the nanoparticle formulation and manufacturing.294 Printing technologies including inkjet printing, particle replication in nonwetting templates (PRINT), and 3D printing are powerful platforms to fabricate microparticles or nanoparticles, which could allow for more control over size, shape, surface chemistry, and mechanical properties.295−297 They facilitate mechanistic

7.3. Manufacturing Considerations for Nonviral Delivery of CRISPR/Cas9

Innovations in materials chemistry will continue to fuel the development of nanodelivery systems for various Cas9/sgRNA configurations. The discussion above has highlighted many innovative chemical designs to form nanoparticles with interesting functionalities, ranging from stealth properties through PEGylation to cell-specific delivery with ligand targeting and to environment-specific unpacking via pHsensitive or bioreducible degradable bonds in the carrier.288,289 However, the formulation or manufacturing of the nanoparticles will be equally important, and this aspect has generally been neglected in the field of nonviral gene delivery. So far, the formation of Cas9/sgRNA nanoparticles is formed by charge neutralization between the cationic gene carriers and the negatively charged Cas9/sgRNA complex via bulk mixing. In research laboratories, it involves pipetting a polycation solution into the DNA/RNA solution in an Eppendorf tube, followed by shaking or more commonly vortexing. Nanoparticles form within milliseconds.290 Although convenient, 9896


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researchers who can make an important contribution by developing effective nonviral and targeted delivery systems for various gene-editing elements.

studies to understand how the nanoparticles’ properties would influence the delivery efficiency, and help establish an accurate property−function relationship. In past years, printing technologies have been used to fabricate nanoparticles for chemical drug, siRNA, and RNA replicon delivery.298−301 However, there are few applications using printing technologies for gene expression plasmid and protein delivery. In addition, the scaleup of microparticles/nanoparticles by print technologies has not been addressed. Thus, CRISPR/Cas9 delivery based on nanocarriers manufactured by top-down technologies remains to be explored. In summary, manufacturing will eventually be a critical barrier for realizing the full potential of nonviral, nanoparticle-mediated Cas9/sgRNA delivery. Technologies that can control nanoparticle characteristics, ensure reproducibility, and facilitate scale-up of the Cas9/sgRNA nanodelivery systems will be needed.

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Mingqiang Li: 0000-0002-5178-4138 Hae-Won Kim: 0000-0001-6400-6100 Gang Bao: 0000-0001-5501-554X Kam W. Leong: 0000-0002-3269-5770 Notes

The authors declare no competing financial interest. Biographies

8. CONCLUDING REMARKS Using a guide RNA and Cas9 nuclease to identify and edit target DNA sequences, the CRISPR/Cas9 system offers advantages over other gene editing technologies with respect to simplicity and versatility. This precise and efficient gene editing tool has continued to evolve rapidly and impacted diverse applications from disease modeling to therapeutic interventions. A clinical trial using CRISPR/Cas9-edited T cells to treat patients with chemotherapy and radiation therapyresistant lung cancer is reported to start.59 Ample opportunities lie ahead, as do challenges for widespread in vivo applications. Meanwhile, concerns for safety persist. The extent of off-target effects should be elucidated, particularly in the context of considering the benefit-to-risk ratio. A clear understanding of the factors contributing to nonspecific DNA binding would be an important first step. Increasing the specificity of CRISPR/ Cas9, such as applying protein engineering and evolution techniques to improve the Cas9 specificity, or using small molecule to turn on/off the Cas9 activity, would be useful. Moreover, engineering gRNA, possibly with the aid of bioinformatics, to modulate the stringency of binding to the DNA target may also improve the specificity. Potential host immune responses caused by Cas9 expression remain to be determined. Limiting to single administration, decreasing the Cas9/gRNA dosage may be a fruitful strategy to minimize their immunogenicity in the future. Delivery is the main challenge for robust implementation of CRISPR/Cas9 gene editing both in vitro and in vivo. The latter would particularly benefit from an effective nonviral vector. Unlike the delivery of Cas9 expression DNA/mRNA, the direct delivery of Cas9 protein into cells provides rapid action, high efficiency, and lower off-target effect. However, nanoparticlemediated delivery of protein is more challenging in many respects than that of nucleic acid therapeutics because of formulation difficulty, from packaging into small size to retention of bioactivity and protection from degradation before entry into the nucleus. The large size of Cas9 protein magnifies the difficulty. There are also fewer choices of carriers for protein, in comparison with nucleic acid. This is a fertile research direction that can advance the CRISPR/Cas9 technology. Targeted delivery via surface decoration of nanoparticles, a topic intensively pursued by many nanomedicine researchers, is another area that would enhance the appeal of this technology. In conclusion, the field of CRISPR/ Cas9 genome editing awaits innovations from interdisciplinary collaboration of biomedical scientists and biomaterials

Hong-Xia Wang obtained her Ph.D. at the University of Science and Technology of China. She is currently a postdoctoral researcher in Prof. Kam Leong’s lab at Columbia University. Her research focuses on the development of nanocarriers for siRNA or CRISPR/Cas9, and in understanding the critical barriers to the systemic administration of the nanomedicines based on siRNA or CRISPR/Cas9. Mingqiang Li received his bachelor’s degree from the University of Science and Technology of China in 2009. He then obtained his Ph.D. degree under the supervision of Prof. Xuesi Chen from the Chinese Academy of Sciences in 2015. He is currently working as a postdoctoral research scientist with Prof. Kam W. Leong at Columbia University. His current research is mainly focused on biomaterials, nanomedicine, and microfluidics. Ciaran Lee received his Ph.D. degree in Molecular Medicine from University College Cork (Cork, Ireland) in 2011. After a postdoctoral fellowship at University College Cork, he moved to the Department of Biomedical Engineering at Georgia Institute of Technology (Georgia) for a Postdoctoral position in Prof. Gang Bao’s lab. In 2015, he took up a position as Director of the genome editing core at Rice University (Texas). His research interests are improving genome editing tools for clinical applications and the development of delivery systems for in vivo gene therapy. Syandan Chakraborty received his medical degree (M.B.B.S) from the Postgraduate Institute of Medical Sciences (Rohtak, India) in 2003, Master’s degree in Bioengineering from Indian Institute of Technology (Kanpur, India) in 2007, and Ph.D. in Biomedical Engineering from Duke University (North Carolina) in 2014. He then moved to the Department of Biomedical Engineering at Columbia University (New York) where he is currently working as a Postdoctoral Research Scientist. His wide-ranging research interests include synthetic biology, cellular reprogramming, genome editing, tissue engineering, and gene delivery. His current research is on generating reprogrammed skeletal myocytes and iPSC-based liver disease models. Hae-Won Kim received his Ph.D. from Seoul National University in 2002. He continued to work on biomaterials and tissue engineering at the Eastman Dental Institute, University College London (UK), with the International Fellowship from Royal Society (2003−2004). After a short period of research in Advanced Materials Institute of SNU, he was appointed as a faculty member at the Dental School of Dankook University (2005). He currently takes the role of director and principal investigator of Institute of Tissue Regeneration Engineering (ITREN, South Korea), and also leads the Department of Nanobiomedical Science and Global Research Center for Regenerative Medicine. His current research is focused on nanobiomaterials, cell−biomaterials 9897


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Mutation Confers Resistance to HIV Infection. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 9591−9596. (14) Wells, D. Gene Therapy Progress and Prospects: Electroporation and Other Physical Methods. Gene Ther. 2004, 11, 1363− 1369. (15) Maurisse, R.; De Semir, D.; Emamekhoo, H.; Bedayat, B.; Abdolmohammadi, A.; Parsi, H.; Gruenert, D. C. Comparative Transfection of DNA into Primary and Transformed Mammalian Cells from Different Lineages. BMC Biotechnol. 2010, 10, 9. (16) Ain, Q. U.; Chung, J. Y.; Kim, Y. H. Current and Future Delivery Systems for Engineered Nucleases: ZFN, TALEN and RGEN. J. Controlled Release 2015, 205, 120−127. (17) Wang, T.; Wei, J. J.; Sabatini, D. M.; Lander, E. S. Genetic Screens in Human Cells Using the CRISPR-Cas9 System. Science 2014, 343, 80−84. (18) Chakraborty, S.; Ji, H.; Kabadi, A. M.; Gersbach, C. A.; Christoforou, N.; Leong, K. W. A CRISPR/Cas9-Based System for Reprogramming Cell Lineage Specification. Stem Cell Rep. 2014, 3, 940−947. (19) Ding, Q.; Strong, A.; Patel, K. M.; Ng, S.-L.; Gosis, B. S.; Regan, S. N.; Cowan, C. A.; Rader, D. J.; Musunuru, K. Permanent Alteration of PCSK9 with in vivo CRISPR-Cas9 Genome Editing. Circ. Res. 2014, 115, 488−492. (20) Ran, F. A.; Cong, L.; Yan, W. X.; Scott, D. A.; Gootenberg, J. S.; Kriz, A. J.; Zetsche, B.; Shalem, O.; Wu, X.; Makarova, K. S.; et al. In vivo Genome Editing Using Staphylococcus Aureus Cas9. Nature 2015, 520, 186−191. (21) Yin, H.; Kanasty, R. L.; Eltoukhy, A. A.; Vegas, A. J.; Dorkin, J. R.; Anderson, D. G. Non-Viral Vectors for Gene-Based Therapy. Nat. Rev. Genet. 2014, 15, 541−555. (22) Liang, X.; Potter, J.; Kumar, S.; Zou, Y.; Quintanilla, R.; Sridharan, M.; Carte, J.; Chen, W.; Roark, N.; Ranganathan, S.; et al. Rapid and Highly Efficient Mammalian Cell Engineering via Cas9 Protein Transfection. J. Biotechnol. 2015, 208, 44−53. (23) Zuris, J. A.; Thompson, D. B.; Shu, Y.; Guilinger, J. P.; Bessen, J. L.; Hu, J. H.; Maeder, M. L.; Joung, J. K.; Chen, Z.-Y.; Liu, D. R. Cationic Lipid-Mediated Delivery of Proteins Enables Efficient Protein-Based Genome Editing in vitro and in vivo. Nat. Biotechnol. 2015, 33, 73−80. (24) Wang, M.; Zuris, J. A.; Meng, F.; Rees, H.; Sun, S.; Deng, P.; Han, Y.; Gao, X.; Pouli, D.; Wu, Q.; et al. Efficient Delivery of Genome-Editing Proteins Using Bioreducible Lipid Nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 2868−2873. (25) Yin, H.; Song, C. Q.; Dorkin, J. R.; Zhu, L. J.; Li, Y.; Wu, Q.; Park, A.; Yang, J.; Suresh, S.; Bizhanova, A.; et al. Therapeutic Genome Editing by Combined Viral and Non-Viral Delivery of CRISPR System Components in vivo. Nat. Biotechnol. 2016, 34, 328−333. (26) Miller, J. B.; Zhang, S.; Kos, P.; Xiong, H.; Zhou, K.; Perelman, S. S.; Zhu, H.; Siegwart, D. J. Non-Viral CRISPR/Cas Gene Editing in vitro and in vivo Enabled by Synthetic Nanoparticle Co-Delivery of Cas9 mRNA and sgRNA. Angew. Chem., Int. Ed. 2017, 56, 1059−1063. (27) Zhen, S.; Hua, L.; Liu, Y.; Gao, L.; Fu, J.; Wan, D.; Dong, L.; Song, H.; Gao, X. Harnessing the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-Associated Cas9 System to Disrupt the Hepatitis B Virus. Gene Ther. 2015, 22, 404− 412. (28) Ebina, H.; Misawa, N.; Kanemura, Y.; Koyanagi, Y. Harnessing the CRISPR/Cas9 System to Disrupt Latent HIV-1 Provirus. Sci. Rep. 2013, 3, 2510. (29) Kennedy, E. M.; Kornepati, A. V.; Goldstein, M.; Bogerd, H. P.; Poling, B. C.; Whisnant, A. W.; Kastan, M. B.; Cullen, B. R. Inactivation of the Human Papillomavirus E6 or E7 Gene in Cervical Carcinoma Cells by Using a Bacterial CRISPR/Cas RNA-Guided Endonuclease. J. Virol. 2014, 88, 11965−11972. (30) Ramakrishna, S.; Dad, A. K.; Beloor, J.; Gopalappa, R.; Lee, S. K.; Kim, H. Gene Disruption by Cell-Penetrating Peptide-Mediated Delivery of Cas9 Protein and Guide RNA. Genome Res. 2014, 24, 1020−1027.

interactions, 3D culture methodologies, and cell reprogramming for musculoskeletal, dental, and neural tissues. Gang Bao is the Foyt Family Professor of Department of Bioengineering and Director of Nanomedicine Center for Nucleoprotein Machines at Rice University. His research focuses on nanomedicine, molecular imaging, and the emerging area of genome editing. He is an elected fellow of the American Institute for Medical and Biological Engineering (2007), the American Physical Society (2007), the American Association for the Advancement of Science (2009), and the American Society of Mechanical Engineers (2009). Kam W. Leong is the Samuel Y. Sheng Professor of Biomedical Engineering at Columbia University, with a joint appointment in the Department of Systems Biology at Columbia University Medical Center. His research focuses on nanoparticle-mediated drug-, gene-, and immuno-therapy, from the design and synthesis of new carriers to applications for cancer, hemophilia, infectious diseases, and cellular reprogramming. He is the Editor-in-Chief of Biomaterials, and a member of the USA National Academy of Engineering.

ACKNOWLEDGMENTS This work is supported by the NIH (HL109442, AI096305, UH3TR000505, and GM110494), the Guangdong Innovative and Entrepreneurial Research Team Program, China (2013S086), and the Global Research Laboratory Program, Korea (2015032163), and the Cancer Prevention and Research Institute of Texas (RR140081). REFERENCES (1) Kay, M. A. State-of-the-Art Gene-Based Therapies: The Road Ahead. Nat. Rev. Genet. 2011, 12, 316−328. (2) Cox, D. B. T.; Platt, R. J.; Zhang, F. Therapeutic Genome Editing: Prospects and Challenges. Nat. Med. 2015, 21, 121−131. (3) Capecchi, M. R. Altering the Genome by Homologous Recombination. Science 1989, 244, 1288−1292. (4) Stoddard, B. L. Homing Endonucleases: From Microbial Genetic Invaders to Reagents for Targeted DNA Modification. Structure 2011, 19, 7−15. (5) Urnov, F. D.; Rebar, E. J.; Holmes, M. C.; Zhang, H. S.; Gregory, P. D. Genome Editing with Engineered Zinc Finger Nucleases. Nat. Rev. Genet. 2010, 11, 636−646. (6) Bogdanove, A. J.; Voytas, D. F. TAL Effectors: Customizable Proteins for DNA Targeting. Science 2011, 333, 1843−1846. (7) Brouns, S. J.; Jore, M. M.; Lundgren, M.; Westra, E. R.; Slijkhuis, R. J.; Snijders, A. P.; Dickman, M. J.; Makarova, K. S.; Koonin, E. V.; Van Der Oost, J. Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes. Science 2008, 321, 960−964. (8) Hsu, P. D.; Lander, E. S.; Zhang, F. Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell 2014, 157, 1262−1278. (9) Wang, M.; Glass, Z.; Xu, Q. Non-Viral Delivery of GenomeEditing Nucleases for Gene Therapy. Gene Ther. 2017, 24, 144. (10) Wu, Y.; Liang, D.; Wang, Y.; Bai, M.; Tang, W.; Bao, S.; Yan, Z.; Li, D.; Li, J. Correction of a Genetic Disease in Mouse via Use of CRISPR-Cas9. Cell Stem Cell 2013, 13, 659−662. (11) Wu, Y.; Zhou, H.; Fan, X.; Zhang, Y.; Zhang, M.; Wang, Y.; Xie, Z.; Bai, M.; Yin, Q.; Liang, D.; et al. Correction of a Genetic Disease by CRISPR-Cas9-Mediated Gene Editing in Mouse Spermatogonial Stem Cells. Cell Res. 2015, 25, 67−79. (12) Long, C.; McAnally, J. R.; Shelton, J. M.; Mireault, A. A.; BasselDuby, R.; Olson, E. N. Prevention of Muscular Dystrophy in Mice by CRISPR/Cas9-Mediated Editing of Germline DNA. Science 2014, 345, 1184−1188. (13) Ye, L.; Wang, J.; Beyer, A. I.; Teque, F.; Cradick, T. J.; Qi, Z.; Chang, J. C.; Bao, G.; Muench, M. O.; Yu, J. Seamless Modification of Wild-Type Induced Pluripotent Stem Cells to the Natural CCR5Δ32 9898


Chemical Reviews

Review

Genome Engineering Using CRISPR/Cas Systems. Science 2013, 339, 819−823. (52) Mali, P.; Yang, L.; Esvelt, K. M.; Aach, J.; Guell, M.; DiCarlo, J. E.; Norville, J. E.; Church, G. M. RNA-Guided Human Genome Engineering via Cas9. Science 2013, 339, 823−826. (53) Hwang, W. Y.; Fu, Y. F.; Reyon, D.; Maeder, M. L.; Tsai, S. Q.; Sander, J. D.; Peterson, R. T.; Yeh, J. R. J.; Joung, J. K. Efficient Genome Editing in Zebrafish Using a CRISPR-Cas System. Nat. Biotechnol. 2013, 31, 227−229. (54) Wang, H.; Yang, H.; Shivalila, C. S.; Dawlaty, M. M.; Cheng, A. W.; Zhang, F.; Jaenisch, R. One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell 2013, 153, 910−918. (55) Niu, Y.; Shen, B.; Cui, Y.; Chen, Y.; Wang, J.; Wang, L.; Kang, Y.; Zhao, X.; Si, W.; Li, W. Generation of Gene-Modified Cynomolgus Monkey Via Cas9/Rna-Mediated Gene Targeting in One-Cell Embryos. Cell 2014, 156, 836−843. (56) Chen, Y.; Zheng, Y.; Kang, Y.; Yang, W.; Niu, Y.; Guo, X.; Tu, Z.; Si, C.; Wang, H.; Xing, R.; et al. Functional Disruption of the Dystrophin Gene in Rhesus Monkey Using CRISPR/Cas9. Hum. Mol. Genet. 2015, 24, 3764−3774. (57) Yin, H.; Xue, W.; Chen, S.; Bogorad, R. L.; Benedetti, E.; Grompe, M.; Koteliansky, V.; Sharp, P. A.; Jacks, T.; Anderson, D. G. Genome Editing with Cas9 in Adult Mice Corrects a Disease Mutation and Phenotype. Nat. Biotechnol. 2014, 32, 551−553. (58) Mullard, A. Novartis Secures First CRISPR Pharma Collaborations. Nat. Rev. Drug Discovery 2015, 14, 82−82. (59) Cyranoski, D. First Trial of CRISPR in People. Nature 2016, 535, 476−477. (60) Hsu, P. D.; Scott, D. A.; Weinstein, J. A.; Ran, F. A.; Konermann, S.; Agarwala, V.; Li, Y.; Fine, E. J.; Wu, X.; Shalem, O. DNA Targeting Specificity of RNA-Guided Cas9 Nucleases. Nat. Biotechnol. 2013, 31, 827−832. (61) Ran, F. A.; Hsu, P. D.; Lin, C.-Y.; Gootenberg, J. S.; Konermann, S.; Trevino, A. E.; Scott, D. A.; Inoue, A.; Matoba, S.; Zhang, Y. Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity. Cell 2013, 154, 1380−1389. (62) Shen, B.; Zhang, W.; Zhang, J.; Zhou, J.; Wang, J.; Chen, L.; Wang, L.; Hodgkins, A.; Iyer, V.; Huang, X.; et al. Efficient Genome Modification by CRISPR-Cas9 Nickase with Minimal Off-Target Effects. Nat. Methods 2014, 11, 399−402. (63) Cho, S. W.; Kim, S.; Kim, Y.; Kweon, J.; Kim, H. S.; Bae, S.; Kim, J. S. Analysis of Off-Target Effects of CRISPR/Cas-Derived RNA-Guided Endonucleases and Nickases. Genome Res. 2014, 24, 132−141. (64) Fu, Y. F.; Sander, J. D.; Reyon, D.; Cascio, V. M.; Joung, J. K. Improving CRISPR-Cas Nuclease Specificity Using Truncated Guide RNAs. Nat. Biotechnol. 2014, 32, 279−284. (65) Qi, L. S.; Larson, M. H.; Gilbert, L. A.; Doudna, J. A.; Weissman, J. S.; Arkin, A. P.; Lim, W. A. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell 2013, 152, 1173−1183. (66) Bikard, D.; Jiang, W.; Samai, P.; Hochschild, A.; Zhang, F.; Marraffini, L. A. Programmable Repression and Activation of Bacterial Gene Expression Using an Engineered CRISPR-Cas System. Nucleic Acids Res. 2013, 41, 7429−7437. (67) Perez-Pinera, P.; Kocak, D. D.; Vockley, C. M.; Adler, A. F.; Kabadi, A. M.; Polstein, L. R.; Thakore, P. I.; Glass, K. A.; Ousterout, D. G.; Leong, K. W.; et al. RNA-Guided Gene Activation by CRISPRCas9-Based Transcription Factors. Nat. Methods 2013, 10, 973−976. (68) Mali, P.; Aach, J.; Stranges, P. B.; Esvelt, K. M.; Moosburner, M.; Kosuri, S.; Yang, L.; Church, G. M. Cas9 Transcriptional Activators for Target Specificity Screening and Paired Nickases for Cooperative Genome Engineering. Nat. Biotechnol. 2013, 31, 833−838. (69) Cheng, A. W.; Wang, H.; Yang, H.; Shi, L.; Katz, Y.; Theunissen, T. W.; Rangarajan, S.; Shivalila, C. S.; Dadon, D. B.; Jaenisch, R. Multiplexed Activation of Endogenous Genes by CRISPR-On, an RNA-Guided Transcriptional Activator System. Cell Res. 2013, 23, 1163−1171.

(31) Platt, R. J.; Chen, S.; Zhou, Y.; Yim, M. J.; Swiech, L.; Kempton, H. R.; Dahlman, J. E.; PaRNAs, O.; Eisenhaure, T. M.; Jovanovic, M.; et al. CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. Cell 2014, 159, 440−455. (32) Mout, R.; Ray, M.; Yesilbag Tonga, G.; Lee, Y. W.; Tay, T.; Sasaki, K.; Rotello, V. M. Direct Cytosolic Delivery of CRISPR/Cas9Ribonucleoprotein for Efficient Gene Editing. ACS Nano 2017, 11, 2452. (33) Li, L.; Song, L.; Liu, X.; Yang, X.; Li, X.; He, T.; Wang, N.; Yang, S.; Yu, C.; Yin, T.; et al. Artificial Virus Delivers CRISPR-Cas9 System for Genome Editing of Cells in Mice. ACS Nano 2017, 11, 95−111. (34) Sun, W.; Ji, W.; Hall, J. M.; Hu, Q.; Wang, C.; Beisel, C. L.; Gu, Z. Self-Assembled DNA Nanoclews for the Efficient Delivery of CRISPR-Cas9 for Genome Editing. Angew. Chem. 2015, 127, 12197− 12201. (35) Doudna, J. A.; Charpentier, E. The New Frontier of Genome Engineering with CRISPR-Cas9. Science 2014, 346, 1258096. (36) Sander, J. D.; Joung, J. K. CRISPR-Cas Systems for Editing, Regulating and Targeting Genomes. Nat. Biotechnol. 2014, 32, 347− 355. (37) Maggio, I.; Goncalves, M. Genome Editing at the Crossroads of Delivery, Specificity, and Fidelity. Trends Biotechnol. 2015, 33, 280− 291. (38) Gori, J. L.; Hsu, P. D.; Maeder, M. L.; Shen, S.; Welstead, G. G.; Bumcrot, D. Delivery and Specificity of CRISPR/Cas9 Genome Editing Technologies for Human Gene Therapy. Hum. Gene Ther. 2015, 26, 443−451. (39) LaFountaine, J. S.; Fathe, K.; Smyth, H. D. C. Delivery and Therapeutic Applications of Gene Editing Technologies ZFNs, TALENs, and CRISPR/Cas9. Int. J. Pharm. 2015, 494, 180−194. (40) Kirchner, M.; Schneider, S. CRISPR-Cas: From the Bacterial Adaptive Immune System to a Versatile Tool for Genome Engineering. Angew. Chem., Int. Ed. 2015, 54, 13508−13514. (41) Singh, A.; Chakraborty, D.; Maiti, S. CRISPR/Cas9: A Historical and Chemical Biology Perspective of Targeted Genome Engineering. Chem. Soc. Rev. 2016, 45, 6666. (42) Hu, J. H.; Davis, K. M.; Liu, D. R. Chemical Biology Approaches to Genome Editing: Understanding, Controlling, and Delivering Programmable Nucleases. Cell Chem. Biol. 2016, 23, 57−73. (43) Ishino, Y.; Shinagawa, H.; Makino, K.; Amemura, M.; Nakata, A. Nucleotide Sequence of the Iap Gene, Responsible for Alkaline Phosphatase Isozyme Conversion in Escherichia Coli, and Identification of the Gene Product. J. Bacteriol. 1987, 169, 5429−5433. (44) Jansen, R.; Embden, J.; Gaastra, W.; Schouls, L. Identification of Genes That Are Associated with DNA Repeats in Prokaryotes. Mol. Microbiol. 2002, 43, 1565−1575. (45) Mojica, F. J.; García-Martínez, J.; Soria, E. Intervening Sequences of Regularly Spaced Prokaryotic Repeats Derive from Foreign Genetic Elements. J. Mol. Evol. 2005, 60, 174−182. (46) Pourcel, C.; Salvignol, G.; Vergnaud, G. CRISPR Elements in Yersinia Pestis Acquire New Repeats by Preferential Uptake of Bacteriophage DNA, and Provide Additional Tools for Evolutionary Studies. Microbiology 2005, 151, 653−663. (47) Bolotin, A.; Quinquis, B.; Sorokin, A.; Ehrlich, S. D. Clustered Regularly Interspaced Short Palindrome Repeats (CRISPRs) Have Spacers of Extrachromosomal Origin. Microbiology 2005, 151, 2551− 2561. (48) Barrangou, R.; Fremaux, C.; Deveau, H.; Richards, M.; Boyaval, P.; Moineau, S.; Romero, D. A.; Horvath, P. CRISPR Provides Acquired Resistance against Viruses in Prokaryotes. Science 2007, 315, 1709−1712. (49) Marraffini, L. A.; Sontheimer, E. J. CRISPR Interference Limits Horizontal Gene Transfer in Staphylococci by Targeting DNA. Science 2008, 322, 1843−1845. (50) Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E. A Programmable Dual-RNA−Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 2012, 337, 816−821. (51) Cong, L.; Ran, F. A.; Cox, D.; Lin, S.; Barretto, R.; Habib, N.; Hsu, P. D.; Wu, X.; Jiang, W.; Marraffini, L. A.; et al. Multiplex 9899


Chemical Reviews

Review

A TALE Nuclease Architecture for Efficient Genome Editing. Nat. Biotechnol. 2011, 29, 143−149. (90) Li, T.; Huang, S.; Jiang, W. Z.; Wright, D.; Spalding, M. H.; Weeks, D. P.; Yang, B. TAL Nucleases (TALNs): Hybrid Proteins Composed of TAL Effectors and FokI DNA-Cleavage Domain. Nucleic Acids Res. 2011, 39, 359−372. (91) Zetsche, B.; Gootenberg, J. S.; Abudayyeh, O. O.; Slaymaker, I. M.; Makarova, K. S.; Essletzbichler, P.; Volz, S. E.; Joung, J.; van der Oost, J.; Regev, A.; et al. Cpf1 Is a Single Rna-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 2015, 163, 759−771. (92) Rosen, L. E.; Morrison, H. A.; Masri, S.; Brown, M. J.; Springstubb, B.; Sussman, D.; Stoddard, B. L.; Seligman, L. M. Homing Endonuclease I-CreI Derivatives with Novel DNA Target Specificities. Nucleic Acids Res. 2006, 34, 4791−4800. (93) Seligman, L. M.; Chevalier, B. S.; Chadsey, M. S.; Edwards, S. T.; Savage, J. H.; Veillet, A. L. Mutations Altering the Cleavage Specificity of a Homing Endonuclease. Nucleic Acids Res. 2002, 30, 3870−3879. (94) Isalan, M. Zinc-Finger Nucleases: How to Play Two Good Hands. Nat. Methods 2012, 9, 32−34. (95) Schmid-Burgk, J. L.; Schmidt, T.; Kaiser, V.; Honing, K.; Hornung, V. A Ligation-Independent Cloning Technique for HighThroughput Assembly of Transcription Activator-Like Effector Genes. Nat. Biotechnol. 2013, 31, 76−81. (96) Holkers, M.; Maggio, I.; Liu, J.; Janssen, J. M.; Miselli, F.; Mussolino, C.; Recchia, A.; Cathomen, T.; Goncalves, M. A. F. V. Differential Integrity of TALE Nuclease Genes Following Adenoviral and Lentiviral Vector Gene Transfer into Human Cells. Nucleic Acids Res. 2013, 41, e63. (97) Cradick, T. J.; Fine, E. J.; Antico, C. J.; Bao, G. CRISPR/Cas9 Systems Targeting Beta-Globin and CCR5 Genes Have Substantial Off-Target Activity. Nucleic Acids Res. 2013, 41, 9584−9592. (98) Fu, Y.; Foden, J. A.; Khayter, C.; Maeder, M. L.; Reyon, D.; Joung, J. K.; Sander, J. D. High-Frequency Off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells. Nat. Biotechnol. 2013, 31, 822−826. (99) Zetsche, B.; Volz, S. E.; Zhang, F. A Split-Cas9 Architecture for Inducible Genome Editing and Transcription Modulation. Nat. Biotechnol. 2015, 33, 139−142. (100) Davis, K. M.; Pattanayak, V.; Thompson, D. B.; Zuris, J. A.; Liu, D. R. Small Molecule−Triggered Cas9 Protein with Improved Genome-Editing Specificity. Nat. Chem. Biol. 2015, 11, 316−318. (101) Fu, Y.; Sander, J. D.; Reyon, D.; Cascio, V. M.; Joung, J. K. Improving CRISPR-Cas Nuclease Specificity Using Truncated Guide RNAs. Nat. Biotechnol. 2014, 32, 279−284. (102) Bolukbasi, M. F.; Gupta, A.; Oikemus, S.; Derr, A. G.; Garber, M.; Brodsky, M. H.; Zhu, L. J.; Wolfe, S. A. DNA-Binding-Domain Fusions Enhance the Targeting Range and Precision of Cas9. Nat. Methods 2015, 12, 1150−1156. (103) Slaymaker, I. M.; Gao, L.; Zetsche, B.; Scott, D. A.; Yan, W. X.; Zhang, F. Rationally Engineered Cas9 Nucleases with Improved Specificity. Science 2016, 351, 84−88. (104) Kleinstiver, B. P.; Pattanayak, V.; Prew, M. S.; Tsai, S. Q.; Nguyen, N. T.; Zheng, Z. L.; Joung, J. K. High-Fidelity CRISPR-Cas9 Nucleases with No Detectable Genome-Wide Off-Target Effects. Nature 2016, 529, 490−495. (105) Smith, C.; Gore, A.; Yan, W.; Abalde-Atristain, L.; Li, Z.; He, C.; Wang, Y.; Brodsky, R. A.; Zhang, K.; Cheng, L.; et al. WholeGenome Sequencing Analysis Reveals High Specificity of CRISPR/ Cas9 and TALEN-Based Genome Editing in Human iPSCs. Cell Stem Cell 2014, 15, 12−13. (106) Suzuki, K.; Yu, C.; Qu, J.; Li, M.; Yao, X.; Yuan, T.; Goebl, A.; Tang, S.; Ren, R.; Aizawa, E.; et al. Targeted Gene Correction Minimally Impacts Whole-Genome Mutational Load in HumanDisease-Specific Induced Pluripotent Stem Cell Clones. Cell Stem Cell 2014, 15, 31−36. (107) Veres, A.; Gosis, B. S.; Ding, Q.; Collins, R.; Ragavendran, A.; Brand, H.; Erdin, S.; Cowan, C. A.; Talkowski, M. E.; Musunuru, K. Low Incidence of Off-Target Mutations in Individual CRISPR-Cas9

(70) Gilbert, L. A.; Larson, M. H.; Morsut, L.; Liu, Z.; Brar, G. A.; Torres, S. E.; Stern-Ginossar, N.; Brandman, O.; Whitehead, E. H.; Doudna, J. A.; et al. CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell 2013, 154, 442−451. (71) Konermann, S.; Brigham, M. D.; Trevino, A. E.; Hsu, P. D.; Heidenreich, M.; Cong, L.; Platt, R. J.; Scott, D. A.; Church, G. M.; Zhang, F. Optical Control of Mammalian Endogenous Transcription and Epigenetic States. Nature 2013, 500, 472−476. (72) Maeder, M. L.; Linder, S. J.; Cascio, V. M.; Fu, Y.; Ho, Q. H.; Joung, J. K. CRISPR RNA-Guided Activation of Endogenous Human Genes. Nat. Methods 2013, 10, 977−979. (73) Konermann, S.; Brigham, M. D.; Trevino, A. E.; Joung, J.; Abudayyeh, O. O.; Barcena, C.; Hsu, P. D.; Habib, N.; Gootenberg, J. S.; Nishimasu, H.; et al. Genome-Scale Transcriptional Activation by an Engineered CRISPR-Cas9 Complex. Nature 2015, 517, 583−588. (74) Zalatan, J. G.; Lee, M. E.; Almeida, R.; Gilbert, L. A.; Whitehead, E. H.; La Russa, M.; Tsai, J. C.; Weissman, J. S.; Dueber, J. E.; Qi, L. S.; et al. Engineering Complex Synthetic Transcriptional Programs with CRISPR RNA Scaffolds. Cell 2015, 160, 339−350. (75) Hilton, I. B.; D’Ippolito, A. M.; Vockley, C. M.; Thakore, P. I.; Crawford, G. E.; Reddy, T. E.; Gersbach, C. A. Epigenome Editing by a CRISPR-Cas9-Based Acetyltransferase Activates Genes from Promoters and Enhancers. Nat. Biotechnol. 2015, 33, 510−517. (76) Thakore, P. I.; Black, J. B.; Hilton, I. B.; Gersbach, C. A. Editing the Epigenome: Technologies for Programmable Transcription and Epigenetic Modulation. Nat. Methods 2016, 13, 127−137. (77) Tsai, S. Q.; Wyvekens, N.; Khayter, C.; Foden, J. A.; Thapar, V.; Reyon, D.; Goodwin, M. J.; Aryee, M. J.; Joung, J. K. Dimeric CRISPR RNA-Guided FokI Nucleases for Highly Specific Genome Editing. Nat. Biotechnol. 2014, 32, 569−576. (78) Guilinger, J. P.; Thompson, D. B.; Liu, D. R. Fusion of Catalytically Inactive Cas9 to FokI Nuclease Improves the Specificity of Genome Modification. Nat. Biotechnol. 2014, 32, 287−305. (79) Wang, S.; Su, J. H.; Feng, Z.; Zhuang, X. An RNA-AptamerBased Two-Color CRISPR Labeling System. Sci. Rep. 2016, 6, 26857. (80) Thierry, A.; Dujon, B. Nested Chromosomal Fragmentation in Yeast Using the Meganuclease I-Sce-I: a New Method for Physical Mapping of Eukaryotic Genomes. Nucleic Acids Res. 1992, 20, 5625− 5631. (81) Smith, J.; Grizot, S.; Arnould, S.; Duclert, A.; Epinat, J. C.; Chames, P.; Prieto, J.; Redondo, P.; Blanco, F. J.; Bravo, J.; et al. A Combinatorial Approach to Create Artificial Homing Endonucleases Cleaving Chosen Sequences. Nucleic Acids Res. 2006, 34, e149. (82) Boissel, S.; Jarjour, J.; Astrakhan, A.; Adey, A.; Gouble, A.; Duchateau, P.; Shendure, J.; Stoddard, B. L.; Certo, M. T.; Baker, D.; et al. MegaTALs: A Rare-Cleaving Nuclease Architecture for Therapeutic Genome Engineering. Nucleic Acids Res. 2014, 42, 2591−2601. (83) Kim, Y. G.; Cha, J.; Chandrasegaran, S. Hybrid Restriction Enzymes: Zinc Finger Fusions to Fok I Cleavage Domain. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 1156−1160. (84) Bibikova, M.; Golic, M.; Golic, K. G.; Carroll, D. Targeted Chromosomal Cleavage and Mutagenesis in Drosophila Using ZincFinger Nucleases. Genetics 2002, 161, 1169−1175. (85) Bibikova, M.; Beumer, K.; Trautman, J. K.; Carroll, D. Enhancing Gene Targeting with Designed Zinc Finger Nucleases. Science 2003, 300, 764−764. (86) Miller, J. C.; Holmes, M. C.; Wang, J.; Guschin, D. Y.; Lee, Y. L.; Rupniewski, I.; Beausejour, C. M.; Waite, A. J.; Wang, N. S.; Kim, K. A.; et al. An Improved Zinc-Finger Nuclease Architecture for Highly Specific Genome Editing. Nat. Biotechnol. 2007, 25, 778−785. (87) Boch, J.; Scholze, H.; Schornack, S.; Landgraf, A.; Hahn, S.; Kay, S.; Lahaye, T.; Nickstadt, A.; Bonas, U. Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors. Science 2009, 326, 1509−1512. (88) Moscou, M. J.; Bogdanove, A. J. A Simple Cipher Governs DNA Recognition by TAL Effectors. Science 2009, 326, 1501−1501. (89) Miller, J. C.; Tan, S. Y.; Qiao, G. J.; Barlow, K. A.; Wang, J. B.; Xia, D. F.; Meng, X. D.; Paschon, D. E.; Leung, E.; Hinkley, S. J.; et al. 9900


Chemical Reviews

Review

and TALEN Targeted Human Stem Cell Clones Detected by WholeGenome Sequencing. Cell Stem Cell 2014, 15, 27−30. (108) Yang, L.; Grishin, D.; Wang, G.; Aach, J.; Zhang, C. Z.; Chari, R.; Homsy, J.; Cai, X.; Zhao, Y.; Fan, J. B.; et al. Targeted and Genome-Wide Sequencing Reveal Single Nucleotide Variations Impacting Specificity of Cas9 in Human Stem Cells. Nat. Commun. 2014, 5, 5507. (109) Siksnys, V.; Gasiunas, G. Rewiring Cas9 to Target New Pam Sequences. Mol. Cell 2016, 61, 793−794. (110) Hirano, S.; Nishimasu, H.; Ishitani, R.; Nureki, O. Structural Basis for the Altered Pam Specificities of Engineered CRISPR-Cas9. Mol. Cell 2016, 61, 886−894. (111) Anders, C.; Bargsten, K.; Jinek, M. Structural Plasticity of PAM Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9. Mol. Cell 2016, 61, 895−902. (112) Makarova, K. S.; Wolf, Y. I.; Alkhnbashi, O. S.; Costa, F.; Shah, S. A.; Saunders, S. J.; Barrangou, R.; Brouns, S. J.; Charpentier, E.; Haft, D. H.; et al. An Updated Evolutionary Classification of CRISPRCas Systems. Nat. Rev. Microbiol. 2015, 13, 722−736. (113) Shmakov, S.; Abudayyeh, O. O.; Makarova, K. S.; Wolf, Y. I.; Gootenberg, J. S.; Semenova, E.; Minakhin, L.; Joung, J.; Konermann, S.; Severinov, K.; et al. Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Mol. Cell 2015, 60, 385−397. (114) Abudayyeh, O. O.; Gootenberg, J. S.; Konermann, S.; Joung, J.; Slaymaker, I. M.; Cox, D. B.; Shmakov, S.; Makarova, K. S.; Semenova, E.; Minakhin, L.; et al. C2c2 Is a Single-Component Programmable RNA-Guided RNA-Targeting CRISPR Effector. Science 2016, 353, aaf5573. (115) Jackson, A. L.; Linsley, P. S. Recognizing and Avoiding siRNA Off-Target Effects for Target Identification and Therapeutic Application. Nat. Rev. Drug Discovery 2010, 9, 57−67. (116) Doench, J. G.; Hartenian, E.; Graham, D. B.; Tothova, Z.; Hegde, M.; Smith, I.; Sullender, M.; Ebert, B. L.; Xavier, R. J.; Root, D. E. Rational Design of Highly Active sgRNAs for CRISPR-Cas9Mediated Gene Inactivation. Nat. Biotechnol. 2014, 32, 1262−1267. (117) Chari, R.; Mali, P.; Moosburner, M.; Church, G. M. Unraveling CRISPR-Cas9 Genome Engineering Parameters via a Library-onLibrary Approach. Nat. Methods 2015, 12, 823−826. (118) Moreno-Mateos, M. A.; Vejnar, C. E.; Beaudoin, J. D.; Fernandez, J. P.; Mis, E. K.; Khokha, M. K.; Giraldez, A. J. CRISPRscan: Designing Highly Efficient sgRNAs for CRISPR-Cas9 Targeting in vivo. Nat. Methods 2015, 12, 982−988. (119) Xu, H.; Xiao, T.; Chen, C. H.; Li, W.; Meyer, C. A.; Wu, Q.; Wu, D.; Cong, L.; Zhang, F.; Liu, J. S.; et al. Sequence Determinants of Improved CRISPR sgRNA Design. Genome Res. 2015, 25, 1147−1157. (120) Wong, N.; Liu, W.; Wang, X. WU-CRISPR: Characteristics of Functional Guide RNAs for the CRISPR/Cas9 System. Genome Biol. 2015, 16, 218. (121) Haeussler, M.; Schonig, K.; Eckert, H.; Eschstruth, A.; Mianne, J.; Renaud, J. B.; Schneider-Maunoury, S.; Shkumatava, A.; Teboul, L.; Kent, J.; et al. Evaluation of Off-Target and On-Target Scoring Algorithms and Integration into the Guide RNA Selection Tool CRISPOR. Genome Biol. 2016, 17, 148. (122) Tsai, S. Q.; Zheng, Z.; Nguyen, N. T.; Liebers, M.; Topkar, V. V.; Thapar, V.; Wyvekens, N.; Khayter, C.; Iafrate, A. J.; Le, L. P.; et al. Guide-Seq Enables Genome-Wide Profiling of Off-Target Cleavage by CRISPR-Cas Nucleases. Nat. Biotechnol. 2015, 33, 187−197. (123) Lin, Y.; Cradick, T. J.; Brown, M. T.; Deshmukh, H.; Ranjan, P.; Sarode, N.; Wile, B. M.; Vertino, P. M.; Stewart, F. J.; Bao, G. CRISPR/Cas9 Systems Have Off-Target Activity with Insertions or Deletions between Target DNA and Guide RNA Sequences. Nucleic Acids Res. 2014, 42, 7473−7485. (124) Singh, R.; Kuscu, C.; Quinlan, A.; Qi, Y.; Adli, M. Cas9Chromatin Binding Information Enables More Accurate CRISPR OffTarget Prediction. Nucleic Acids Res. 2015, 43, e118. (125) Montague, T. G.; Cruz, J. M.; Gagnon, J. A.; Church, G. M.; Valen, E. CHOPCHOP: A CRISPR/Cas9 and TALEN Web Tool for Genome Editing. Nucleic Acids Res. 2014, 42, W401−407.

(126) Stemmer, M.; Thumberger, T.; Del Sol Keyer, M.; Wittbrodt, J.; Mateo, J. L. CCTop: An Intuitive, Flexible and Reliable CRISPR/ Cas9 Target Prediction Tool. PLoS One 2015, 10, e0124633. (127) Zhu, L. J.; Holmes, B. R.; Aronin, N.; Brodsky, M. H. CRISPRseek: A Bioconductor Package to Identify Target-Specific Guide RNAs for CRISPR-Cas9 Genome-Editing Systems. PLoS One 2014, 9, e108424. (128) Heigwer, F.; Kerr, G.; Boutros, M. E-CRISP: Fast CRISPR Target Site Identification. Nat. Methods 2014, 11, 122−123. (129) Gratz, S. J.; Ukken, F. P.; Rubinstein, C. D.; Thiede, G.; Donohue, L. K.; Cummings, A. M.; O’Connor-Giles, K. M. Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila. Genetics 2014, 196, 961−971. (130) O’Brien, A.; Bailey, T. L. GT-Scan: Identifying Unique Genomic Targets. Bioinformatics 2014, 30, 2673−2675. (131) Xie, S.; Shen, B.; Zhang, C.; Huang, X.; Zhang, Y. sgRNAcas9: A Software Package for Designing CRISPR sgRNA and Evaluating Potential Off-Target Cleavage Sites. PLoS One 2014, 9, e100448. (132) Hodgkins, A.; Farne, A.; Perera, S.; Grego, T.; Parry-Smith, D. J.; Skarnes, W. C.; Iyer, V. WGE: A CRISPR Database for Genome Engineering. Bioinformatics 2015, 31, 3078−3080. (133) Cradick, T. J.; Qui, P.; Lee, C. M.; Fine, E. J.; Bao, G. COSMID: A Web-Based Tool for Identifying and Validating CRISPR/ Cas Off-Target Sites. Mol. Ther.–Nucleic Acids 2014, 3, e214. (134) Bae, S.; Park, J.; Kim, J. S. Cas-OFFinder: A Fast and Versatile Algorithm That Searches for Potential Off-Target Sites of Cas9 RNAGuided Endonucleases. Bioinformatics 2014, 30, 1473−1475. (135) Sander, J. D.; Zaback, P.; Joung, J. K.; Voytas, D. F.; Dobbs, D. Zinc Finger Targeter (ZiFiT): An Engineered Zinc Finger/Target Site Design Tool. Nucleic Acids Res. 2007, 35, W599−W605. (136) Kim, D.; Bae, S.; Park, J.; Kim, E.; Kim, S.; Yu, H. R.; Hwang, J.; Kim, J. I.; Kim, J. S. Digenome-Seq: Genome-Wide Profiling of CRISPR-Cas9 Off-Target Effects in Human Cells. Nat. Methods 2015, 12, 237−243. (137) Wang, X.; Wang, Y.; Wu, X.; Wang, J.; Qiu, Z.; Chang, T.; Huang, H.; Lin, R. J.; Yee, J. K. Unbiased Detection of Off-Target Cleavage by CRISPR-Cas9 and TALENs Using Integrase-Defective Lentiviral Vectors. Nat. Biotechnol. 2015, 33, 175−178. (138) Kuscu, C.; Arslan, S.; Singh, R.; Thorpe, J.; Adli, M. GenomeWide Analysis Reveals Characteristics of Off-Target Sites Bound by the Cas9 Endonuclease. Nat. Biotechnol. 2014, 32, 677−683. (139) Kleinstiver, B. P.; Prew, M. S.; Tsai, S. Q.; Nguyen, N. T.; Topkar, V. V.; Zheng, Z.; Joung, J. K. Broadening the Targeting Range of Staphylococcus Aureus CRISPR-Cas9 by Modifying PAM Recognition. Nat. Biotechnol. 2015, 33, 1293−1298. (140) Friedland, A. E.; Baral, R.; Singhal, P.; Loveluck, K.; Shen, S.; Sanchez, M.; Marco, E.; Gotta, G. M.; Maeder, M. L.; Kennedy, E. M.; et al. Characterization of Staphylococcus Aureus Cas9: A Smaller Cas9 for All-in-One Adeno-Associated Virus Delivery and Paired Nickase Applications. Genome Biol. 2015, 16, 257. (141) O’Geen, H.; Henry, I. M.; Bhakta, M. S.; Meckler, J. F.; Segal, D. J. A Genome-Wide Analysis of Cas9 Binding Specificity Using ChIP-Seq and Targeted Sequence Capture. Nucleic Acids Res. 2015, 43, 3389−3404. (142) Wu, X.; Scott, D. A.; Kriz, A. J.; Chiu, A. C.; Hsu, P. D.; Dadon, D. B.; Cheng, A. W.; Trevino, A. E.; Konermann, S.; Chen, S.; et al. Genome-Wide Binding of the CRISPR Endonuclease Cas9 in Mammalian Cells. Nat. Biotechnol. 2014, 32, 670−676. (143) Frock, R. L.; Hu, J.; Meyers, R. M.; Ho, Y. J.; Kii, E.; Alt, F. W. Genome-Wide Detection of DNA Double-Stranded Breaks Induced by Engineered Nucleases. Nat. Biotechnol. 2015, 33, 179−186. (144) Crosetto, N.; Mitra, A.; Silva, M. J.; Bienko, M.; Dojer, N.; Wang, Q.; Karaca, E.; Chiarle, R.; Skrzypczak, M.; Ginalski, K.; et al. Nucleotide-Resolution DNA Double-Strand Break Mapping by NextGeneration Sequencing. Nat. Methods 2013, 10, 361−365. (145) Sternberg, S. H.; LaFrance, B.; Kaplan, M.; Doudna, J. A. Conformational Control of DNA Target Cleavage by CRISPR-Cas9. Nature 2015, 527, 110−113. 9901


Chemical Reviews

Review

(146) Kiani, S.; Chavez, A.; Tuttle, M.; Hall, R. N.; Chari, R.; TerOvanesyan, D.; Qian, J.; Pruitt, B. W.; Beal, J.; Vora, S.; et al. Cas9 gRNA Engineering for Genome Editing, Activation and Repression. Nat. Methods 2015, 12, 1051−1054. (147) Xue, W.; Chen, S.; Yin, H.; Tammela, T.; Papagiannakopoulos, T.; Joshi, N. S.; Cai, W.; Yang, G.; Bronson, R.; Crowley, D. G.; et al. CRISPR-Mediated Direct Mutation of Cancer Genes in the Mouse Liver. Nature 2014, 514, 380−384. (148) Choi, P. S.; Meyerson, M. Targeted Genomic Rearrangements Using CRISPR/Cas Technology. Nat. Commun. 2014, 5, 3728. (149) Matano, M.; Date, S.; Shimokawa, M.; Takano, A.; Fujii, M.; Ohta, Y.; Watanabe, T.; Kanai, T.; Sato, T. Modeling Colorectal Cancer Using CRISPR-Cas9-Mediated Engineering of Human Intestinal Organoids. Nat. Med. 2015, 21, 256−262. (150) Zuckermann, M.; Hovestadt, V.; Knobbe-Thomsen, C. B.; Zapatka, M.; Northcott, P. A.; Schramm, K.; Belic, J.; Jones, D. T.; Tschida, B.; Moriarity, B.; et al. Somatic CRISPR/Cas9-Mediated Tumour Suppressor Disruption Enables Versatile Brain Tumour Modelling. Nat. Commun. 2015, 6, 7391. (151) Heckl, D.; Kowalczyk, M. S.; Yudovich, D.; Belizaire, R.; Puram, R. V.; McConkey, M. E.; Thielke, A.; Aster, J. C.; Regev, A.; Ebert, B. L. Generation of Mouse Models of Myeloid Malignancy with Combinatorial Genetic Lesions Using CRISPR-Cas9 Genome Editing. Nat. Biotechnol. 2014, 32, 941−946. (152) Carroll, K. J.; Makarewich, C. A.; Mcanally, J.; Anderson, D. M.; Zentilin, L.; Liu, N.; Giacca, M.; Basselduby, R.; Olson, E. N. A Mouse Model for Adult Cardiac-Specific Gene Deletion with CRISPR/Cas9. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 338−343. (153) Ma, Y.; Shen, B.; Zhang, X.; Lu, Y.; Chen, W.; Ma, J.; Huang, X.; Zhang, L. Heritable Multiplex Genetic Engineering in Rats Using CRISPR/Cas9. PLoS One 2014, 9, e89413. (154) Whitworth, K. M.; Lee, K.; Benne, J. A.; Beaton, B. P.; Spate, L. D.; Murphy, S. L.; Samuel, M. S.; Mao, J.; O’Gorman, C.; Walters, E. M.; et al. Use of the CRISPR/Cas9 System to Produce Genetically Engineered Pigs from in vitro-Derived Oocytes and Embryos. Biol. Reprod. 2014, 91, 78. (155) Horii, T.; Tamura, D.; Morita, S.; Kimura, M.; Hatada, I. Generation of an ICF Syndrome Model by Efficient Genome Editing of Human Induced Pluripotent Stem Cells Using the CRISPR System. Int. J. Mol. Sci. 2013, 14, 19774−19781. (156) Paquet, D.; Kwart, D.; Chen, A.; Sproul, A.; Jacob, S.; Teo, S.; Olsen, K. M.; Gregg, A.; Noggle, S.; Tessierlavigne, M. Efficient Introduction of Specific Homozygous and Heterozygous Mutations Using CRISPR/Cas9. Nature 2016, 533, 125−129. (157) Schwank, G.; Koo, B.-K.; Sasselli, V.; Dekkers, J. F.; Heo, I.; Demircan, T.; Sasaki, N.; Boymans, S.; Cuppen, E.; van der Ent, C. K.; et al. Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients. Cell stem cell 2013, 13, 653−658. (158) Long, C.; Amoasii, L.; Mireault, A. A.; McAnally, J. R.; Li, H.; Sanchez-Ortiz, E.; Bhattacharyya, S.; Shelton, J. M.; Bassel-Duby, R.; Olson, E. N. Postnatal Genome Editing Partially Restores Dystrophin Expression in a Mouse Model of Muscular Dystrophy. Science 2016, 351, 400−403. (159) Tabebordbar, M.; Zhu, K.; Cheng, J. K.; Chew, W. L.; Widrick, J. J.; Yan, W. X.; Maesner, C.; Wu, E. Y.; Xiao, R.; Ran, F. A.; et al. In vivo Gene Editing in Dystrophic Mouse Muscle and Muscle Stem Cells. Science 2016, 351, 407−411. (160) Nelson, C. E.; Hakim, C. H.; Ousterout, D. G.; Thakore, P. I.; Moreb, E. A.; Rivera, R. M. C.; Madhavan, S.; Pan, X.; Ran, F. A.; Yan, W. X.; et al. In vivo Genome Editing Improves Muscle Function in a Mouse Model of Duchenne Muscular Dystrophy. Science 2016, 351, 403−407. (161) Liu, Y.; Zeng, Y.; Liu, L.; Zhuang, C.; Fu, X.; Huang, W.; Cai, Z. Synthesizing and Gate Genetic Circuits Based on CRISPR-Cas9 for Identification of Bladder Cancer Cells. Nat. Commun. 2014, 5, 5393. (162) Antal, C. E.; Hudson, A. M.; Kang, E.; Zanca, C.; Wirth, C.; Stephenson, N. L.; Trotter, E. W.; Gallegos, L. L.; Miller, C. J.; Furnari,

F. B.; et al. Cancer-Associated Protein Kinase C Mutations Reveal Kinase’s Role as Tumor Suppressor. Cell 2015, 160, 489−502. (163) Kaminski, R.; Chen, Y.; Fischer, T.; Tedaldi, E.; Napoli, A.; Zhang, Y.; Karn, J.; Hu, W.; Khalili, K. Elimination of HIV-1 Genomes from Human T-Lymphoid Cells by CRISPR/Cas9 Gene Editing. Sci. Rep. 2016, 6, 22555. (164) Kaminski, R.; Bella, R.; Yin, C.; Otte, J.; Ferrante, P.; Gendelman, H.; Li, H.; Booze, R.; Gordon, J.; Hu, W.; et al. Excision of HIV-1 DNA by Gene Editing: A Proof-of-Concept in vivo Study. Gene Ther. 2016, 23, 690−695. (165) Dong, C.; Qu, L.; Wang, H.; Wei, L.; Dong, Y.; Xiong, S. Targeting Hepatitis B Virus cccDNA by CRISPR/Cas9 Nuclease Efficiently Inhibits Viral Replication. Antiviral Res. 2015, 118, 110− 117. (166) Price, A. A.; Sampson, T. R.; Ratner, H. K.; Grakoui, A.; Weiss, D. S. Cas9-Mediated Targeting of Viral RNA in Eukaryotic Cells. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 6164−6169. (167) Roehm, P. C.; Shekarabi, M.; Wollebo, H. S.; Bellizzi, A.; He, L.; Salkind, J.; Khalili, K. Inhibition of HSV-1 Replication by Gene Editing Strategy. Sci. Rep. 2016, 6, 23146. (168) Wang, J.; Quake, S. R. RNA-Guided Endonuclease Provides a Therapeutic Strategy to Cure Latent Herpesviridae Infection. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 13157−13162. (169) O’Connell, M. R.; Oakes, B. L.; Sternberg, S. H.; East-Seletsky, A.; Kaplan, M.; Doudna, J. A. Programmable RNA Recognition and Cleavage by CRISPR/Cas9. Nature 2014, 516, 263−266. (170) Nelles, D. A.; Fang, M. Y.; Aigner, S.; Yeo, G. W. Applications of Cas9 as an RNA-Programmed RNA-Binding Protein. BioEssays 2015, 37, 732−739. (171) Sampson, T. R.; Saroj, S. D.; Llewellyn, A. C.; Tzeng, Y.-L.; Weiss, D. S. A CRISPR/Cas System Mediates Bacterial Innate Immune Evasion and Virulence. Nature 2013, 497, 254−257. (172) Tsai, S. Q.; Joung, J. K. Defining and Improving the GenomeWide Specificities of CRISPR-Cas9 Nucleases. Nat. Rev. Genet. 2016, 17, 300−312. (173) Chew, W. L.; Tabebordbar, M.; Cheng, J. K.; Mali, P.; Wu, E. Y.; Ng, A. H.; Zhu, K.; Wagers, A. J.; Church, G. M. A Multifunctional AAV-CRISPR-Cas9 and Its Host Response. Nat. Methods 2016, 13, 868−874. (174) Wang, D.; Mou, H.; Li, S.; Li, Y.; Hough, S.; Tran, K.; Li, J.; Yin, H.; Anderson, D. G.; Sontheimer, E. J.; et al. AdenovirusMediated Somatic Genome Editing of PTEN by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses. Hum. Gene Ther. 2015, 26, 432−442. (175) Ran, F. A.; Hsu, P. D.; Wright, J.; Agarwala, V.; Scott, D. A.; Zhang, F. Genome Engineering Using the CRISPR-Cas9 System. Nat. Protoc. 2013, 8, 2281−2308. (176) Chen, S.; Sanjana, N. E.; Zheng, K.; Shalem, O.; Lee, K.; Shi, X.; Scott, D. A.; Song, J.; Pan, J. Q.; Weissleder, R.; et al. GenomeWide CRISPR Screen in a Mouse Model of Tumor Growth and Metastasis. Cell 2015, 160, 1246−1260. (177) Hemmi, H.; Takeuchi, O.; Kawai, T.; Kaisho, T.; Sato, S.; Sanjo, H.; Matsumoto, M.; Hoshino, K.; Wagner, H.; Takeda, K.; et al. A Toll-Like Receptor Recognizes Bacterial DNA. Nature 2000, 408, 740−745. (178) Han, X.; Liu, Z.; Chan, Jo. M.; Zhang, K.; Li, Y.; Zeng, Z.; Li, N.; Zu, Y.; Qin, L. CRISPR-Cas9 Delivery to Hard-to-Transfect Cells via Membrane Deformation. Sci. Adv. 2015, 1, e1500454. (179) Ousterout, D. G.; Kabadi, A. M.; Thakore, P. I.; Majoros, W. H.; Reddy, T. E.; Gersbach, C. A. Multiplex CRISPR/Cas9-Based Genome Editing for Correction of Dystrophin Mutations That Cause Duchenne Muscular Dystrophy. Nat. Commun. 2015, 6, 6244. (180) Sakuma, T.; Nishikawa, A.; Kume, S.; Chayama, K.; Yamamoto, T. Multiplex Genome Engineering in Human Cells Using All-in-One CRISPR/Cas9 Vector System. Sci. Rep. 2015, 4, 5400. (181) Yang, H.; Wang, H.; Shivalila, C. S.; Cheng, A. W.; Shi, L.; Jaenisch, R. One-Step Generation of Mice Carrying Reporter and 9902


Chemical Reviews

Review

(202) Yang, H.; Wang, H. Y.; Shivalila, C. S.; Cheng, A. W.; Shi, L. Y.; Jaenisch, R. One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering. Cell 2013, 154, 1370−1379. (203) Yang, D.; Xu, J.; Zhu, T.; Fan, J.; Lai, L.; Zhang, J.; Chen, Y. E. Effective Gene Targeting in Rabbits Using RNA-Guided Cas9 Nucleases. J. Mol. Cell Biol. 2014, 6, 97−99. (204) Crispo, M.; Mulet, A.; Tesson, L.; Barrera, N.; Cuadro, F.; dos Santos-Neto, P.; Nguyen, T.; Crénéguy, A.; Brusselle, L.; Anegon, I.; et al. Efficient Generation of Myostatin Knock-out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes. PLoS One 2015, 10, e0136690. (205) Goodwin, T.; Huang, L. Nonviral Vectors: We Have Come a Long Way. Adv. Genet. 2014, 88, 1−12. (206) Lin, S.-R.; Yang, H.-C.; Kuo, Y.-T.; Liu, C.-J.; Yang, T.-Y.; Sung, K.-C.; Lin, Y.-Y.; Wang, H.-Y.; Wang, C.-C.; Shen, Y.-C.; et al. The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates in vivo. Mol. Ther.–Nucleic Acids 2014, 3, e186. (207) Tlaxca, J. L.; Rychak, J. J.; Ernst, P. B.; Konkalmatt, P. R.; Shevchenko, T. I.; Pizzaro, T. T.; Rivera-Nieves, J.; Klibanov, A. L.; Lawrence, M. B. Ultrasound-Based Molecular Imaging and Specific Gene Delivery to Mesenteric Vasculature by Endothelial Adhesion Molecule Targeted Microbubbles in a Mouse Model of Crohn’s Disease. J. Controlled Release 2013, 165, 216−225. (208) Sun, L.; Huang, C.-W.; Wu, J.; Chen, K.-J.; Li, S.-H.; Weisel, R. D.; Rakowski, H.; Sung, H.-W.; Li, R.-K. The Use of Cationic Microbubbles to Improve Ultrasound-Targeted Gene Delivery to the Ischemic Myocardium. Biomaterials 2013, 34, 2107−2116. (209) Lin, C.-Y.; Hsieh, H.-Y.; Pitt, W. G.; Huang, C.-Y.; Tseng, I.-C.; Yeh, C.-K.; Wei, K.-C.; Liu, H.-L. Focused Ultrasound-Induced BloodBrain Barrier Opening for Non-Viral, Non-Invasive, and Targeted Gene Delivery. J. Controlled Release 2015, 212, 1−9. (210) Tirlapur, U. K.; König, K. Cell Biology: Targeted Transfection by Femtosecond Laser. Nature 2002, 418, 290−291. (211) Sharei, A.; Zoldan, J.; Adamo, A.; Sim, W. Y.; Cho, N.; Jackson, E.; Mao, S.; Schneider, S.; Han, M.-J.; Lytton-Jean, A.; et al. A VectorFree Microfluidic Platform for Intracellular Delivery. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 2082−2087. (212) Wightman, L.; Kircheis, R.; Rössler, V.; Carotta, S.; Ruzicka, R.; Kursa, M.; Wagner, E. Different Behavior of Branched and Linear Polyethylenimine for Gene Delivery in vitro and in vivo. J. Gene Med. 2001, 3, 362−372. (213) Godbey, W.; Wu, K. K.; Mikos, A. G. Size Matters: Molecular Weight Affects the Efficiency of Poly (Ethyleneimine) as a Gene Delivery Vehicle. J. Biomed. Mater. Res. 1999, 45, 268−275. (214) Buscail, L.; Bournet, B.; Vernejoul, F.; Cambois, G.; Lulka, H.; Hanoun, N.; Dufresne, M.; Meulle, A.; Vignolle-Vidoni, A.; Ligat, L.; et al. First-in-Man Phase 1 Clinical Trial of Gene Therapy for Advanced Pancreatic Cancer: Safety, Biodistribution, and Preliminary Clinical Findings. Mol. Ther. 2015, 23, 779−789. (215) Hanna, N.; Ohana, P.; Konikoff, F.; Leichtmann, G.; Hubert, A.; Appelbaum, L.; Kopelman, Y.; Czerniak, A.; Hochberg, A. Phase 1/ 2a, Dose-Escalation, Safety, Pharmacokinetic and Preliminary Efficacy Study of Intratumoral Administration of BC-819 in Patients with Unresectable Pancreatic Cancer. Cancer Gene Ther. 2012, 19, 374− 381. (216) Francis, S. M.; Taylor, C. A.; Tang, T.; Liu, Z.; Zheng, Q.; Dondero, R.; Thompson, J. E. SNS01-T Modulation of eIF5A Inhibits B-Cell Cancer Progression and Synergizes with Bortezomib and Lenalidomide. Mol. Ther. 2014, 22, 1643−1652. (217) Dash, M.; Chiellini, F.; Ottenbrite, R. M.; Chiellini, E. Chitosan-a Versatile Semi-Synthetic Polymer in Biomedical Applications. Prog. Polym. Sci. 2011, 36, 981−1014. (218) Kiang, T.; Wen, J.; Lim, H. W.; Leong, K. W. The Effect of the Degree of Chitosan Deacetylation on the Efficiency of Gene Transfection. Biomaterials 2004, 25, 5293−5301. (219) Huang, M.; Fong, C.-W.; Khor, E.; Lim, L.-Y. Transfection Efficiency of Chitosan Vectors: Effect of Polymer Molecular Weight and Degree of Deacetylation. J. Controlled Release 2005, 106, 391−406.

Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering. Cell 2013, 154, 1370−1379. (182) Kim, S.; Kim, D.; Cho, S. W.; Kim, J.; Kim, J.-S. Highly Efficient RNA-Guided Genome Editing in Human Cells via Delivery of Purified Cas9 Ribonucleoproteins. Genome Res. 2014, 24, 1012− 1019. (183) Schumann, K.; Lin, S.; Boyer, E.; Simeonov, D. R.; Subramaniam, M.; Gate, R. E.; Haliburton, G. E.; Chun, J. Y.; Bluestone, J. A.; Doudna, J. A.; et al. Generation of Knock-in Primary Human T Cells Using Cas9 Ribonucleoproteins. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 10437−10442. (184) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2, 751−760. (185) Wisse, E.; Jacobs, F.; Topal, B.; Frederik, P.; De Geest, B. The Size of Endothelial Fenestrae in Human Liver Sinusoids: Implications for Hepatocyte-Directed Gene Transfer. Gene Ther. 2008, 15, 1193− 1199. (186) D’Astolfo, D. S.; Pagliero, R. J.; Pras, A.; Karthaus, W. R.; Clevers, H.; Prasad, V.; Lebbink, R. J.; Rehmann, H.; Geijsen, N. Efficient Intracellular Delivery of Native Proteins. Cell 2015, 161, 674−690. (187) Shalem, O.; Sanjana, N. E.; Hartenian, E.; Shi, X.; Scott, D. A.; Mikkelsen, T. S.; Heckl, D.; Ebert, B. L.; Root, D. E.; Doench, J. G.; et al. Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Science 2014, 343, 84−87. (188) Koike-Yusa, H.; Li, Y.; Tan, E.-P.; Velasco-Herrera, M. D. C.; Yusa, K. Genome-Wide Recessive Genetic Screening in Mammalian Cells with a Lentiviral CRISPR-Guide RNA Library. Nat. Biotechnol. 2014, 32, 267−273. (189) Mátrai, J.; Chuah, M. K.; VandenDriessche, T. Recent Advances in Lentiviral Vector Development and Applications. Mol. Ther. 2010, 18, 477−490. (190) Chen, X.; Goncalves, M. A. F. V. Engineered Viruses as Genome Editing Devices. Mol. Ther. 2016, 24, 447−457. (191) Kotterman, M. A.; Schaffer, D. V. Engineering AdenoAssociated Viruses for Clinical Gene Therapy. Nat. Rev. Genet. 2014, 15, 445−451. (192) Kotterman, M. A.; Chalberg, T. W.; Schaffer, D. V. Viral Vectors for Gene Therapy: Translational and Clinical Outlook. Annu. Rev. Biomed. Eng. 2015, 17, 63−89. (193) McCarty, D. M.; Young, S. M., Jr.; Samulski, R. J. Integration of Adeno-Associated Virus (AAV) and Recombinant AAV Vectors. Annu. Rev. Genet. 2004, 38, 819−845. (194) Wu, Z.; Yang, H.; Colosi, P. Effect of Genome Size on AAV Vector Packaging. Mol. Ther. 2010, 18, 80−86. (195) Esvelt, K. M.; Mali, P.; Braff, J. L.; Moosburner, M.; Yaung, S. J.; Church, G. M. Orthogonal Cas9 Proteins for RNA-Guided Gene Regulation and Editing. Nat. Methods 2013, 10, 1116−1121. (196) Swiech, L.; Heidenreich, M.; Banerjee, A.; Habib, N.; Li, Y.; Trombetta, J.; Sur, M.; Zhang, F. In vivo Interrogation of Gene Function in the Mammalian Brain Using CRISPR-Cas9. Nat. Biotechnol. 2015, 33, 102−106. (197) Truong, D. J. J.; Kuhner, K.; Kuhn, R.; Werfel, S.; Engelhardt, S.; Wurst, W.; Ortiz, O. Development of an Intein-Mediated SplitCas9 System for Gene Therapy. Nucleic Acids Res. 2015, 43, 6450− 6458. (198) Pack, D. W.; Hoffman, A. S.; Pun, S.; Stayton, P. S. Design and Development of Polymers for Gene Delivery. Nat. Rev. Drug Discovery 2005, 4, 581−593. (199) Mintzer, M. A.; Simanek, E. E. Nonviral Vectors for Gene Delivery. Chem. Rev. 2008, 109, 259−302. (200) Graessmann, M.; Graessmann, A. Microinjection of Tissue Culture Cells. Methods Enzymol. 1983, 101, 482−492. (201) Kimura, Y.; Hisano, Y.; Kawahara, A.; Higashijima, S.-i. Efficient Generation of Knock-in Transgenic Zebrafish Carrying Reporter/Driver Genes by CRISPR/Cas9-Mediated Genome Engineering. Sci. Rep. 2015, 4, 6545. 9903


Chemical Reviews

Review

Nonviral Delivery of Self-Amplifying RNA Vaccines. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 14604−14609. (238) Kauffman, K. J.; Dorkin, J. R.; Yang, J. H.; Heartlein, M. W.; DeRosa, F.; Mir, F. F.; Fenton, O. S.; Anderson, D. G. Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in vivo with Fractional Factorial and Definitive Screening Designs. Nano Lett. 2015, 15, 7300−7306. (239) Mahiny, A. J.; Dewerth, A.; Mays, L. E.; Alkhaled, M.; Mothes, B.; Malaeksefat, E.; Loretz, B.; Rottenberger, J.; Brosch, D. M.; Reautschnig, P.; et al. In vivo Genome Editing Using NucleaseEncoding mRNA Corrects SP-B Deficiency. Nat. Biotechnol. 2015, 33, 584−586. (240) Kallen, K.-J.; Heidenreich, R.; Schnee, M.; Petsch, B.; Schlake, T.; Thess, A.; Baumhof, P.; Scheel, B.; Koch, S. D.; Fotin-Mleczek, M. A Novel, Disruptive Vaccination Technology: Self-Adjuvanted RNActive® Vaccines. Hum. Vaccines Immunother. 2013, 9, 2263− 2276. (241) Fotin-Mleczek, M.; Duchardt, K. M.; Lorenz, C.; Pfeiffer, R.; Ojkic-Zrna, S.; Probst, J.; Kallen, K.-J. Messenger RNA-Based Vaccines with Dual Activity Induce Balanced TLR-7 Dependent Adaptive Immune Responses and Provide Antitumor Activity. J. Immunother. 2011, 34, 1−15. (242) Ü zgün, S.; Nica, G.; Pfeifer, C.; Bosinco, M.; Michaelis, K.; Lutz, J.-F.; Schneider, M.; Rosenecker, J.; Rudolph, C. Pegylation Improves Nanoparticle Formation and Transfection Efficiency of Messenger RNA. Pharm. Res. 2011, 28, 2223−2232. (243) Phua, K. K.; Leong, K. W.; Nair, S. K. Transfection Efficiency and Transgene Expression Kinetics of mRNA Delivered in Naked and Nanoparticle Format. J. Controlled Release 2013, 166, 227−233. (244) Weide, B.; Pascolo, S.; Scheel, B.; Derhovanessian, E.; Pflugfelder, A.; Eigentler, T. K.; Pawelec, G.; Hoerr, I.; Rammensee, H.-G.; Garbe, C. Direct Injection of Protamine-Protected mRNA: Results of a Phase 1/2 Vaccination Trial in Metastatic Melanoma Patients. J. Immunother. 2009, 32, 498−507. (245) Hendel, A.; Bak, R. O.; Clark, J. T.; Kennedy, A. B.; Ryan, D. E.; Roy, S.; Steinfeld, I.; Lunstad, B. D.; Kaiser, R. J.; Wilkens, A. B.; et al. Chemically Modified Guide RNAs Enhance CRISPR-Cas Genome Editing in Human Primary Cells. Nat. Biotechnol. 2015, 33, 985−989. (246) Lu, Y.; Sun, W.; Gu, Z. Stimuli-Responsive Nanomaterials for Therapeutic Protein Delivery. J. Controlled Release 2014, 194, 1−19. (247) Gu, Z.; Biswas, A.; Zhao, M.; Tang, Y. Tailoring Nanocarriers for Intracellular Protein Delivery. Chem. Soc. Rev. 2011, 40, 3638− 3655. (248) Paix, A.; Folkmann, A.; Rasoloson, D.; Seydoux, G. High Efficiency, Homology-Directed Genome Editing in Caenorhabditis Elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes. Genetics 2015, 201, 47−54. (249) Woo, J. W.; Kim, J.; Kwon, S. I.; Corvalan, C.; Cho, S. W.; Kim, H.; Kim, S. G.; Kim, S. T.; Choe, S.; Kim, J. S. DNA-Free Genome Editing in Plants with Preassembled CRISPR-Cas9 Ribonucleoproteins. Nat. Biotechnol. 2015, 33, 1162−1164. (250) Cho, S. W.; Lee, J.; Carroll, D.; Kim, J.-S.; Lee, J. Heritable Gene Knockout in Caenorhabditis Elegans by Direct Injection of Cas9−sgRNA Ribonucleoproteins. Genetics 2013, 195, 1177−1180. (251) Roberts, M. J.; Bentley, M. D.; Harris, J. M. Chemistry for Peptide and Protein Pegylation. Adv. Drug Delivery Rev. 2012, 64, 116−127. (252) Alconcel, S. N. S.; Baas, A. S.; Maynard, H. D. FDA-Approved Poly(Ethylene Glycol)-Protein Conjugate Drugs. Polym. Chem. 2011, 2, 1442−1448. (253) Cárdenas-Bailón, F.; Osorio-Revilla, G.; Gallardo-Velázquez, T. Microencapsulation of Insulin Using a W/O/W Double Emulsion Followed by Complex Coacervation to Provide Protection in the Gastrointestinal Tract. J. Microencapsulation 2015, 32, 1−9. (254) Davis, M. E. The First Targeted Delivery of siRNA in Humans via a Self-Assembling, Cyclodextrin Polymer-Based Nanoparticle: From Concept to Clinic. Mol. Pharmaceutics 2009, 6, 659−668.

(220) Kim, Y. H.; Gihm, S. H.; Park, C. R.; Lee, K. Y.; Kim, T. W.; Kwon, I. C.; Chung, H.; Jeong, S. Y. Structural Characteristics of SizeControlled Self-Aggregates of Deoxycholic Acid-Modified Chitosan and Their Application as a DNA Delivery Carrier. Bioconjugate Chem. 2001, 12, 932−938. (221) Xu, X.; Capito, R. M.; Spector, M. Plasmid Size Influences Chitosan Nanoparticle Mediated Gene Transfer to Chondrocytes. J. Biomed. Mater. Res., Part A 2008, 84, 1038−1048. (222) Oliveira, A. V. V; Silva, G. A.; Chung, D. C. Enhancement of Chitosan-Mediated Gene Delivery through Combination with PhiC31 Integrase. Acta Biomater. 2015, 17 (17), 89−97. (223) Konstan, M. W.; Davis, P. B.; Wagener, J. S.; Hilliard, K. A.; Stern, R. C.; Milgram, L. J. H.; Kowalczyk, T. H.; Hyatt, S. L.; Fink, T. L.; Gedeon, C. R.; et al. Compacted DNA Nanoparticles Administered to the Nasal Mucosa of Cystic Fibrosis Subjects Are Safe and Demonstrate Partial to Complete Cystic Fibrosis Transmembrane Regulator Reconstitution. Hum. Gene Ther. 2004, 15, 1255−1269. (224) Fink, T.; Klepcyk, P.; Oette, S.; Gedeon, C.; Hyatt, S.; Kowalczyk, T.; Moen, R.; Cooper, M. Plasmid Size up to 20 Kbp Does Not Limit Effective in vivo Lung Gene Transfer Using Compacted DNA Nanoparticles. Gene Ther. 2006, 13, 1048−1051. (225) Allen, T. M.; Cullis, P. R. Liposomal Drug Delivery Systems: From Concept to Clinical Applications. Adv. Drug Delivery Rev. 2013, 65, 36−48. (226) Luo, D.; Saltzman, W. M. Enhancement of Transfection by Physical Concentration of DNA at the Cell Surface. Nat. Biotechnol. 2000, 18, 893−895. (227) Ghosh, P.; Han, G.; De, M.; Kim, C. K.; Rotello, V. M. Gold Nanoparticles in Delivery Applications. Adv. Drug Delivery Rev. 2008, 60, 1307−1315. (228) Bates, K.; Kostarelos, K. Carbon Nanotubes as Vectors for Gene Therapy: Past Achievements, Present Challenges and Future Goals. Adv. Drug Delivery Rev. 2013, 65, 2023−2033. (229) Wang, T.; Upponi, J. R.; Torchilin, V. P. Design of Multifunctional Non-Viral Gene Vectors to Overcome Physiological Barriers: Dilemmas and Strategies. Int. J. Pharm. 2012, 427, 3−20. (230) Kogure, K.; Moriguchi, R.; Sasaki, K.; Ueno, M.; Futaki, S.; Harashima, H. Development of a Non-Viral Multifunctional EnvelopeType Nano Device by a Novel Lipid Film Hydration Method. J. Controlled Release 2004, 98, 317−323. (231) Nakamura, T.; Akita, H.; Yamada, Y.; Hatakeyama, H.; Harashima, H. A Multifunctional Envelope-Type Nanodevice for Use in Nanomedicine: Concept and Applications. Acc. Chem. Res. 2012, 45, 1113−1121. (232) Kormann, M. S.; Hasenpusch, G.; Aneja, M. K.; Nica, G.; Flemmer, A. W.; Herber-Jonat, S.; Huppmann, M.; Mays, L. E.; Illenyi, M.; Schams, A.; et al. Expression of Therapeutic Proteins after Delivery of Chemically Modified mRNA in Mice. Nat. Biotechnol. 2011, 29, 154−157. (233) Thess, A.; Grund, S.; Mui, B. L.; Hope, M. J.; Baumhof, P.; Fotin-Mleczek, M.; Schlake, T. Sequence-Engineered mRNA without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals. Mol. Ther. 2015, 23, 1456−1464. (234) Karikó, K.; Muramatsu, H.; Keller, J. M.; Weissman, D. Increased Erythropoiesis in Mice Injected with Submicrogram Quantities of Pseudouridine-Containing mRNA Encoding Erythropoietin. Mol. Ther. 2012, 20, 948−953. (235) Pollard, C.; Rejman, J.; De Haes, W.; Verrier, B.; Van Gulck, E.; Naessens, T.; De Smedt, S.; Bogaert, P.; Grooten, J.; Vanham, G.; et al. Type I IFN Counteracts the Induction of Antigen-Specific Immune Responses by Lipid-Based Delivery of mRNA Vaccines. Mol. Ther. 2013, 21, 251−259. (236) Rejman, J.; Tavernier, G.; Bavarsad, N.; Demeester, J.; De Smedt, S. C. mRNA Transfection of Cervical Carcinoma and Mesenchymal Stem Cells Mediated by Cationic Carriers. J. Controlled Release 2010, 147, 385−391. (237) Geall, A. J.; Verma, A.; Otten, G. R.; Shaw, C. A.; Hekele, A.; Banerjee, K.; Cu, Y.; Beard, C. W.; Brito, L. A.; Krucker, T.; et al. 9904


Chemical Reviews

Review

(275) Peacock, H.; Kannan, A.; Beal, P. A.; Burrows, C. J. Chemical Modification of siRNA Bases to Probe and Enhance RNA Interference. J. Org. Chem. 2011, 76, 7295−7300. (276) Bramsen, J. B.; Laursen, M. B.; Nielsen, A. F.; Hansen, T. B.; Bus, C.; Langkjaer, N.; Babu, B. R.; Hojland, T.; Abramov, M.; Van Aerschot, A.; et al. A Large-Scale Chemical Modification Screen Identifies Design Rules to Generate siRNAs with High Activity, High Stability and Low Toxicity. Nucleic Acids Res. 2009, 37, 2867−2881. (277) Pottier, N.; Cauffiez, C.; Perrais, M.; Barbry, P.; Mari, B. FibromiRs: Translating Molecular Discoveries into New Anti-Fibrotic Drugs. Trends Pharmacol. Sci. 2014, 35, 119−126. (278) Meyers, B. C.; Green, P. J. Plant MicroRNAs: Methods and Protocols; Humana Press: Totowa, NJ, 2010; p 137. (279) Lennox, K. A.; Behlke, M. A. Chemical Modification and Design of Anti-miRNA Oligonucleotides. Gene Ther. 2011, 18, 1111− 1120. (280) Peng, B.; Chen, Y.; Leong, K. W. MicroRNA Delivery for Regenerative Medicine. Adv. Drug Delivery Rev. 2015, 88, 108−122. (281) Li, Z.; Rana, T. M. Therapeutic Targeting of MicroRNAs: Current Status and Future Challenges. Nat. Rev. Drug Discovery 2014, 13, 622−638. (282) Lao, Y. H.; Phua, K. K.; Leong, K. W. Aptamer Nanomedicine for Cancer Therapeutics: Barriers and Potential for Translation. ACS Nano 2015, 9, 2235−2254. (283) Rusconi, C. P.; Scardino, E.; Layzer, J.; Pitoc, G. A.; Ortel, T. L.; Monroe, D.; Sullenger, B. A. RNA Aptamers as Reversible Antagonists of Coagulation Factor IXa. Nature 2002, 419, 90−94. (284) Dellinger, D. J.; Sheehan, D. M.; Christensen, N. K.; Lindberg, J. G.; Caruthers, M. H. Solid-Phase Chemical Synthesis of Phosphonoacetate and Thiophosphonoacetate Oligodeoxynucleotides. J. Am. Chem. Soc. 2003, 125, 940−950. (285) Sharma, V. K.; Sharma, R. K.; Singh, S. K. Antisense Oligonucleotides: Modifications and Clinical Trials. MedChemComm 2014, 5, 1454−1471. (286) Wagner, E. Biomaterials in RNAi Therapeutics: Quo Vadis? Biomater. Sci. 2013, 1, 804−809. (287) Gagnon, K. T.; Corey, D. R. Stepping toward Therapeutic CRISPR. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 15536−15537. (288) Putnam, D. Polymers for Gene Delivery Across Length Scales. Nat. Mater. 2006, 5, 439−451. (289) Williford, J. M.; Wu, J.; Ren, Y.; Archang, M. M.; Leong, K. W.; Mao, H. Q. Recent Advances in Nanoparticle-Mediated Sirna Delivery. Annu. Rev. Biomed. Eng. 2014, 16, 347−370. (290) Ho, Y. P.; Chen, H. H.; Leong, K. W.; Wang, T. H. The Convergence of Quantum-Dot-Mediated Fluorescence Resonance Energy Transfer and Microfluidics for Monitoring DNA Polyplex Self-Assembly in Real Time. Nanotechnology 2009, 20, 095103. (291) Ho, Y. P.; Grigsby, C. L.; Zhao, F.; Leong, K. W. Tuning Physical Properties of Nanocomplexes through Microfluidics-Assisted Confinement. Nano Lett. 2011, 11, 2178−2182. (292) Grigsby, C. L.; Ho, Y. P.; Lin, C.; Engbersen, J. F.; Leong, K. W. Microfluidic Preparation of Polymer-Nucleic Acid Nanocomplexes Improves Nonviral Gene Transfer. Sci. Rep. 2013, 3, 3155. (293) Lu, M.; Ho, Y. P.; Grigsby, C. L.; Nawaz, A. A.; Leong, K. W.; Huang, T. J. Three-Dimensional Hydrodynamic Focusing Method for Polyplex Synthesis. ACS Nano 2014, 8, 332−339. (294) Valencia, P. M.; Farokhzad, O. C.; Karnik, R.; Langer, R. Microfluidic Technologies for Accelerating the Clinical Translation of Nanoparticles. Nat. Nanotechnol. 2012, 7, 623−629. (295) Perry, J. L.; Herlihy, K. P.; Napier, M. E.; Desimone, J. M. Print: A Novel Platform toward Shape and Size Specific Nanoparticle Theranostics. Acc. Chem. Res. 2011, 44, 990−998. (296) Campbell, T. A.; Ivanova, O. S. 3d Printing of Multifunctional Nanocomposites. Nano Today 2013, 8, 119−120. (297) Tekin, E.; Smith, P. J.; Schubert, U. S. Inkjet Printing as a Deposition and Patterning Tool for Polymers and Inorganic Particles. Soft Matter 2008, 4, 703−713. (298) Dunn, S. S.; Tian, S. M.; Blake, S.; Wang, J.; Galloway, A. L.; Murphy, A.; Pohlhaus, P. D.; Rolland, J. P.; Napier, M. E.; DeSimone,

(255) Zimmermann, T. S.; Lee, A. C.; Akinc, A.; Bramlage, B.; Bumcrot, D.; Fedoruk, M. N.; Harborth, J.; Heyes, J. A.; Jeffs, L. B.; John, M.; et al. RNAi-Mediated Gene Silencing in Non-Human Primates. Nature 2006, 441, 111−114. (256) Kontermann, R. E. Strategies for Extended Serum Half-Life of Protein Therapeutics. Curr. Opin. Biotechnol. 2011, 22, 868−876. (257) Rahdar, M.; McMahon, M. A.; Prakash, T. P.; Swayze, E. E.; Bennett, C. F.; Cleveland, D. W. Synthetic CRISPR RNA-Cas9Guided Genome Editing in Human Cells. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, E7110−E7117. (258) Scheule, R. K.; George, J. A. S.; Bagley, R. G.; Marshall, J.; Kaplan, J. M.; Akita, G. Y.; Wang, K. X.; Lee, E. R.; Harris, D. J.; Jiang, C.; et al. Basis of Pulmonary Toxicity Associated with Cationic LipidMediated Gene Transfer to the Mammalian Lung. Hum. Gene Ther. 1997, 8, 689−707. (259) Li, S.; Tseng, W.; Stolz, D. B.; Wu, S.; Watkins, S.; Huang, L. Dynamic Changes in the Characteristics of Cationic Lipidic Vectors after Exposure to Mouse Serum: Implications for Intravenous Lipofection. Gene Ther. 1999, 6, 585−594. (260) Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. S. Poly (Ethylene Glycol) in Drug Delivery: Pros and Cons as Well as Potential Alternatives. Angew. Chem., Int. Ed. 2010, 49, 6288−6308. (261) McManus, J. J.; Rädler, J. O.; Dawson, K. A. Observation of a Rectangular Columnar Phase in a DNA-Calcium-Zwitterionic Lipid Complex. J. Am. Chem. Soc. 2004, 126, 15966−15967. (262) Rodriguez, P. L.; Harada, T.; Christian, D. A.; Pantano, D. A.; Tsai, R. K.; Discher, D. E. Minimal ″Self″ Peptides That Inhibit Phagocytic Clearance and Enhance Delivery of Nanoparticles. Science 2013, 339, 971−975. (263) Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.; Terada, Y.; Kano, M.; Miyazono, K.; Uesaka, M.; et al. Accumulation of Sub-100 nm Polymeric Micelles in Poorly Permeable Tumours Depends on Size. Nat. Nanotechnol. 2011, 6, 815−823. (264) Li, L.; Wang, H.; Ong, Z. Y.; Xu, K.; Ee, P. L. R.; Zheng, S.; Hedrick, J. L.; Yang, Y.-Y. Polymer-and Lipid-Based Nanoparticle Therapeutics for the Treatment of Liver Diseases. Nano Today 2010, 5, 296−312. (265) Schratzberger, P.; Krainin, J. G.; Schratzberger, G.; Silver, M.; Ma, H.; Kearney, M.; Zuk, R. F.; Brisken, A. F.; Losordo, D. W.; Isner, J. M. Transcutaneous Ultrasound Augments Naked DNA Transfection of Skeletal Muscle. Mol. Ther. 2002, 6, 576−583. (266) Capecchi, M. R. High Efficiency Transformation by Direct Microinjection of DNA into Cultured Mammalian Cells. Cell 1980, 22, 479−488. (267) Brandén, L. J.; Mohamed, A. J.; Smith, C. E. A Peptide Nucleic Acid-Nuclear Localization Signal Fusion That Mediates Nuclear Transport of DNA. Nat. Biotechnol. 1999, 17, 784−787. (268) Schaffer, D. V.; Fidelman, N. A.; Dan, N.; Lauffenburger, D. A. Vector Unpacking as a Potential Barrier for Receptor-Mediated Polyplex Gene Delivery. Biotechnol. Bioeng. 2000, 67, 598−606. (269) Benner, S. A.; Kim, H. J.; Carrigan, M. A. Asphalt, Water, and the Prebiotic Synthesis of Ribose, Ribonucleosides, and RNA. Acc. Chem. Res. 2012, 45, 2025−2034. (270) Deleavey, G. F.; Damha, M. J. Designing Chemically Modified Oligonucleotides for Targeted Gene Silencing. Chem. Biol. 2012, 19, 937−954. (271) Braasch, D. A.; Jensen, S.; Liu, Y.; Kaur, K.; Arar, K.; White, M. A.; Corey, D. R. RNA Interference in Mammalian Cells by ChemicallyModified RNA. Biochemistry 2003, 42, 7967−7975. (272) Sundaram, P.; Kurniawan, H.; Byrne, M. E.; Wower, J. Therapeutic RNA Aptamers in Clinical Trials. Eur. J. Pharm. Sci. 2013, 48, 259−271. (273) Jordheim, L. P.; Durantel, D.; Zoulim, F.; Dumontet, C. Advances in the Development of Nucleoside and Nucleotide Analogues for Cancer and Viral Diseases. Nat. Rev. Drug Discovery 2013, 12, 447−464. (274) Kanasty, R.; Dorkin, J. R.; Vegas, A.; Anderson, D. Delivery Materials for siRNA Therapeutics. Nat. Mater. 2013, 12, 967−977. 9905


Chemical Reviews

Review

J. M. Reductively Responsive siRNA-Conjugated Hydrogel Nanoparticles for Gene Silencing. J. Am. Chem. Soc. 2012, 134, 7423−7430. (299) Petros, R. A.; Ropp, P. A.; DeSimone, J. M. Reductively Labile Print Particles for the Delivery of Doxorubicin to Hela Cells. J. Am. Chem. Soc. 2008, 130, 5008. (300) Hasan, W.; Chu, K.; Gullapalli, A.; Dunn, S. S.; Enlow, E. M.; Luft, J. C.; Tian, S. M.; Napier, M. E.; Pohlhaus, P. D.; Rolland, J. P.; et al. Delivery of Multiple siRNAs Using Lipid-Coated PLGA Nanoparticles for Treatment of Prostate Cancer. Nano Lett. 2012, 12, 287−292. (301) Xu, J.; Luft, J. C.; Yi, X. W.; Tian, S. M.; Owens, G.; Wang, J.; Johnson, A.; Berglund, P.; Smith, J.; Napier, M. E.; et al. RNA Replicon Delivery Via Lipid-Complexed Print Protein Particles. Mol. Pharmaceutics 2013, 10, 3366−3374.

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