Emerging Approaches for Spatiotemporal Control of Targeted

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Emerging approaches for spatiotemporal control of targeted genome with inducible CRISPR-Cas9 Yuta Nihongaki, Takahiro Otabe, and Moritoshi Sato Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04757 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 22, 2017

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Analytical Chemistry

Emerging approaches for spatiotemporal control of targeted genome with inducible CRISPR-Cas9

Yuta Nihongaki1,2, Takahiro Otabe1 and Moritoshi Sato1,* 1

Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan.

2

Present address: Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

*To whom correspondence should be addressed. Tel: +81-3-5454-6579; Fax: +81-3-5454-6579; E-mail: [email protected]

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The breakthrough CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nuclease has revolutionized our ability in genome engineering. Although Cas9 is already a powerful tool for simple and efficient target endogenous gene manipulation, further engineering of Cas9 will improve the performance of Cas9, such as gene-editing efficiency and accuracy in vivo, and expand the application possibility of this Cas9 technology. The emerging inducible Cas9 methods, which can control the activity of Cas9 using an external stimulus such as chemicals and light, have the potential to provide spatiotemporal gene manipulation in user-defined cell population at a specific time and improve the accuracy of Cas9-mediated genome editing. In this review, we focus on the recent advance in inducible Cas9 technologies, especially light-inducible Cas9, and related methodologies, and also discuss future directions of this emerging tools.

Introduction of genome engineering Gene targeting Understanding the gene functions has been greatly facilitated by the technologies enabling to manipulate DNA sequences in endogenous genomic contexts. The technique termed gene targeting was reported in yeast over 40 years ago and it is still popular and powerful technique for editing the DNA sequence of specific gene1. The scope of gene targeting was rapidly expanded to higher organisms. Modifying genomic sequence of mouse embryonic stem cells leads to generate mice having desired genomic mutations, which are useful for investigating gene functions in vivo and creating model mice of human diseases. Gene targeting requires the exogenous DNA donor which has an arbitrary DNA sequence flanked with 5’- and 3’-overhang complementary DNA sequences to a desired integration locus. When this DNA donor is introduced into cells, it could be integrated into complementary endogenous genome via homologous recombination with low frequency, which overwrites endogenous genome with defined DNA sequence. Gene targeting enables to remove specific regions on genome, add desired 2 ACS Paragon Plus Environment

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exogenous DNA sequence into targeted region and introduce point mutations. Furthermore, the development of Cre-loxP recombination system considerably contribute to the improvement of gene targeting technology by offering developmental stage and/or organ specific gene manipulation in living mice2, which have expanded our ability to manipulate and understand genome. However, gene targeting was often expensive, time-consuming and labor-intensive procedure and applicable to only some organisms because the rate of successful exogenous DNA integration into targeted locus is extremely low3.

Genome editing with programmable nucleases The emerging technology, named genome editing, has greatly improved the efficiency of gene targeting, offering the inexpensive and easy approach for manipulating endogenous DNA sequence in diverse cell lines and species. Genome editing is facilitated by an artificially inducing DNA double-strand break (DSB) in a targeted gene (Figure 1A)4. In genome editing, programmable DNA nucleases are used to induce DSB in the specific genomic region. In eukaryotic cells, DSB is repaired by cellular endogenous mechanisms, nonhomologous end-joining (NHEJ) pathway and homology-directed repair (HDR) pathway. In most cases, DSB is repaired by NHEJ pathway. NHEJ repair pathway is error-prone, so that small deletions or insertions are introduced in the repaired targeted gene. Because genes have information for protein synthesis in the form of triplet DNA code, the NHEJ-mediated indel mutation induces DNA frame-shift and disturb the correct translation, resulting in the failure of functional protein translation called gene knockout. In addition, genome editing also offers precise gene modification with higher efficiency than conventional gene targeting. In contrast to NHEJ-mediated error-prone repair pathway, HDR pathway offers precise error-free gene modification. In HDR process, DSB is repaired with reference to template sequences. By introducing the homologous exogenous DNA template containing point mutations or exogenous gene, the targeted gene in the genome can be mutated precisely and can be tagged with exogenous proteins.



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Programmable DNA nuclease is key to successful genome editing. There are three kinds of programmable DNA nucleases, zinc finger nucleases (ZFN)5, transcription activator-like effector nucleases (TALEN)6–8, and Cas99–12. ZFNs consist of three repeated zinc finger DNA-binding units and a FokI DNA-cleavage domain (Figure 1B). One zinc finger units recognize three base pair of DNA, and thus three repeated zinc finger units can bind to a specific nine base pair of DNA. Because a FokI DNAcleavage domain can work only when it dimerizes, a pair of ZFNs directed to the neighboring sequences of target site can recognize a specific eighteen base pair DNA sequence and induce a DSB at the targeted locus, facilitating genome modification. TALENs also consist of repeated DNA-binding units and a DNA-cleavage domain (Figure 1C). Unlike ZFNs, one TALE units recognize one base pair of DNA and its sequence specificity is higher than ZFNs. Both ZFNs and TALENs used repeated protein for targeting DNA sequence, and therefore scientists who want to edit their target gene have to reassembly DNA constructs encoding repeated protein units that can bind to and cleave their target sequence, which process is time-consuming and requires specialized expertise. Compared to ZFNs and TALENs, Cas9 offers more simple approach for genome editing13 (Figure 1D). Cas9 is one of the proteins compose a bacterial CRISPR immune system14,15. In the CRISPR immunity, Cas9 plays an important role that digests an invader’s DNA using crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as a guide RNA. Harnessing this RNA-guided DNA cleavage by Cas9, several groups independently reported Cas9 can be used for facilitating genome engineering in mammalian cells9–12. In general, genome engineering with Cas9 requires introduction of two components, Cas9 protein and single guide RNA (sgRNA), a fusion RNA of crRNA and tracrRNA9. Cas9 protein is complexed with sgRNA and the Cas9:sgRNA complex can bind to and cleave a DNA sequence that is complementary to the first 20 nucleotides of an sgRNA. Therefore, just modifying the first 20 nucleotides of sgRNA, Cas9 can be targeted to an arbitrary DNA sequence. Although Cas9 can only target DNA sequences adjacent to a protospacer-adjacent motif (PAM), the PAM of Streptococcus pyogenes Cas9


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(SpCas9), which is widely used in genome editing at present, is simple and abundant anywhere (5’-NGG3’), and thus SpCas9 can target any desired gene.

Spatiotemporal control of genome sequence Chemically-inducible Cas9 Because Cas9-based genome editing has high efficiency and scalability, it enables the invention of unprecedented applications including genome-wide screening16–19 and in vivo gene editing20–24. In addition to these development of Cas9 applications, engineering of Cas9 molecules has been also exploited, which could further expand the application possibility of Cas9 technology. In particular, inducible Cas9 is one of the most exciting field of engineered Cas9. Recently, diverse chemicallyinducible Cas9s have been developed, which enable spatial and temporal activation of Cas9 in a small molecule dependent manner. By using chemically-inducible Cas9, temporal drug treatment would confine the Cas9 activation within a specific developmental stage for avoiding lethal toxicity and genetic compensation. Also, controlling the duration of Cas9 activation using drugs has the potential to reduce off-target genome modification caused by constitutively active Cas9. The simplest way for controlling Cas9 activity with drugs is regulating the transcription of Cas9. It was demonstrated that by combining Cas9 with doxycycline-inducible transcription system Cas9 expression can be controlled in a doxycycline-dependent manner, and this system enables the induction of genome editing in adult mice21. Similar system is also implemented into human pluripotent stem cells25. In addition, doxycyclineinducible sgRNA systems have been also developed for regulating Cas9-mediated genome regulation26,27. For example, the chimeric promoter sequence based on H1 promoter and doxycycline-inducible promoter offers chemically-inducible sgRNA transcription27. In contrast to transcription control of Cas9 and sgRNA, various chemically-inducible Cas9s based on post-translational regulation have been also exploited, which could offer faster activation and deactivation of Cas9. The rapamycin-inducible Cas9 is 5 ACS Paragon Plus Environment


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based on split fragments of SpCas9 fused to FKBP (FK506 binding protein 12) and FRB (FKBP12 rapamycin binding domain) (Figure 2A)28. Rapamycin treatment induces heterodimerization of FKBP and FRB, subsequently resulting in recomplementation of split Cas9 fragments. Tamoxifen-inducible Cas9 has been also developed using a tamoxifen-inducible intein (Figure 2B)29. In this system, the inducible intein is inserted in Cas9, and it inactivates Cas9. Tamoxifen treatment activates the inserted intein, and the protein splicing mediated by the activated intein restores functional Cas9. Using this inducible intein-Cas9, they demonstrated that reducing the duration of Cas9 activation could improve the accuracy of genome editing. There is another strategy for regulating Cas9 activity in a tamoxifeninducible manner (Figure 2C)30,31. The hormone-binding domain of the estrogen receptor (ERT2) is localized in cytoplasm, and it translocates into nucleus upon tamoxifen treatment. Then Cas9 fused with ERT2 was sequestered in cytoplasm, and in the presence of tamoxifen enter nucleus where Cas9 can work. There are also methods allowing temporal control of Cas9 activity harnessing conditional Cas9 destabilization32,33 (Figure 2D). Cas9 fused with FKBP12-derived destabilizing domain (DD-Cas9) enables temporal control of Cas9 with an FKBP12 synthetic ligand Shield-134. Lastly, a rapidly chemically inducible Cas9 (ciCas9) has been recently developed, which can induce indel mutations in mammalian cells within 2 h35 (Figure 2E). ciCas9 is a single-polypeptide Cas9 variant engineered by replacing REC2 domain of Cas9 with the BCL-xL proteins and fusing an BH3 peptide to the C-terminus of Cas9. A3, a compound that inhibits the interaction between BCL-xL and BH3, can activate ciCas9 within minutes. In addition to these methods based on post-translational control of Cas9, there are also several methods for post-transcriptional control of sgRNA, which can induce Cas9-mediated gene manipulation with drugs36,37. Furthermore, the Cas9 regulation based on bio-orthogonal chemical reaction was also exploited. Luo et al. developed a small molecule switch for controlling protein activity via phosphine-mediated Staudinger reduction38. Using artificial transfer RNA synthetase and tRNA, they introduced ortho-azidobenzyloxycarbonyl amino acid into position K866 of Cas9, which regulates conformational change when Cas9:sgRNA complex forms. This chemically engineered Cas9 is rendered inactive, and small-molecule phosphine can activate it. 6 ACS Paragon Plus Environment

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Light-inducible Cas9 While methods of chemically-inducible Cas9 have been extensively exploited, the examples of light-inducible Cas9 are fewer. In contrast to chemicals, light is a more ideal inducer because of high spatiotemporal resolution and noninvasiveness. A light-activated Cas9 was reported by the site-specific incorporation of a photocaged lysine amino acid39. Ultraviolet illumination can uncage the photocaged Cas9 in living cells, which induces genome editing. The approach based on photocaged molecule is also applied to the engineering of sgRNAs, enabling optical control of Cas9 activity40. A photocleavable ssDNA oligonucleotide, which is complementary to the target region of the guide RNA, prevents an sgRNA from binding to the target DNA and when this protector photocleavable oligo is photolyzed by UV irradiation, the sgRNA is released to bind to and cleave the target DNA. These photocaged approaches are based on the chemical modification of DNA or protein, which requires special expertise and has fundamental difficulty in the delivery of caged product to living organisms. For the last decade, the optogenetic approaches offer light-inducible control of biological activity in vivo without chemical modification41,42. By applying optogenetic technology into Cas9, our group developed an engineered photoactivatable Cas9 (paCas9), which consists of natural amino acids, enabling optical control of Cas9mediated genome editing in human cells43 (Figure 3A). paCas9 consists of two split fragments of Cas9 fused with light-inducible dimerization domains named Magnet44. The Magnet system developed by our group is paired photoswitchable proteins, named positive Magnet (pMag) and negative Magnet (nMag). In the dark, split fragments of Cas9 are inactivated. Upon blue light illumination, pMag and nMag heterodimerize and subsequently split Cas9 fragments are reassembled, resulting in the regain of Cas9 activity. This paCas9 can induce genome editing in HEK293T cells via NHEJ-mediated and HDRmediated pathways. Using spatially-confined blue light irradiation, paCas9 can be spatially activated, and the activity of paCas9 can be switched off by simply turning off the light. Recently, another optogenetic Cas9 tool for light-inducible control of gene editing has been reported, which is named photoswitchable Cas9 (ps-Cas9)45 (Figure 3B). In contrast to paCas9 consisting of the two chimeric constructs based on



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split Cas9 fragments, ps-Cas9 employs a single-polypeptide architecture. In ps-Cas9, the photodissociable dimeric fluorescent protein pdDronpa1 is inserted into the REC2 and PI domains of Cas9, respectively46. In the dark, the activity of ps-Cas9 is sterically inhibited by the dimerization of the inserted pdDronpa1. By 500 nm illumination, pdDronpa1 dissociates and enables Cas9 to bind to and cut a target DNA. There is also another engineered light-switchable Cas9, a fusion protein of SpCas9 and Rhodobacter sphaeroides LOV domain (RsLOV)47 (Figure 3C). In the dark state, RsLOV forms homodimers and blue-light absorption promotes dissociation of RsLOV dimer. Then Cas9 fused to RsLOV also forms homodimer and this dimerization sterically inhibits the Cas9 activity in the dark. Blue light exposure promotes the dissociation of Cas9-RsLOV2 dimer, and increase the functional Cas9-RsLOV2 monomer. It was demonstrated that Cas9-RsLOV2 has light-inducible genome cleavage activity in E. coli, but it is not determined whether Cas9-RsLOV2 works in eukaryotic cells.

Other light-inducible genome engineering technologies In addition to light-inducible CRISPR-Cas9 systems as we listed above, other light-inducible genome engineering technologies which have distinct advantages are available. In particular, methods of optogenetic Cre-loxP recombination have been developed48–51. Although Cre-mediated genome engineering requires the pre-integration of artificial loxP sequences in target region, Cre enables sophisticated genome manipulation such as insertion and deletion of desired DNA sequence and inversion of targeted DNA2. By fusing Arabidopsis thaliana cryptochrome 2 (CRY2) and its binding partner CIB1, which can heterodimerize in response to blue light, to split Cre fragments respectively, light-inducible DNA recombination in living cells has been firstly demonstrated48. This group subsequently showed that this system can be expressed in the mouse brain and regulate gene expression using optical fibers or a two-photon microscope49 and the dynamic range of the photoactivatable Cre recombinase has been also improved through engineering CRY2-CIB1 dimerization system50. Our group has also developed an improved photoactivatable Cre recombinase based on the Magnet dimerization system51. This improved photoactivatable Cre shows higher recombination efficiency than the previously described 8 ACS Paragon Plus Environment

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photoactivatable Cre and can induce DNA recombination in mice liver with blue light. In contrast to the light-inducible genome engineering tools based on Cas9 and Cre which have high sequence-specificity, an optogenetic tool for non-specific random mutagenesis is also available. A histone-fused miniSOG, the mini singlet oxygen generator, can induces broad variation of mutation from single nucleotide mutation to chromosomal deletion in C. elegans52, which is useful for forward genetic screening.

Inducible nuclease-dead Cas9 for precisely controlling gene expression Nuclease-dead Cas9 technologies for manipulating gene transcription By engineering nuclease-dead Cas9, the applications of CRISPR have been extremely expanded beyond genome editing53. The nuclease-dead SpCas9 (SpCas9 having D10A and H840A mutations, hereafter called dCas9) can bind to, but not cleave targeted DNA sequence and dCas9 can be applied to sequence-specific transcriptional repression, termed CRISPR interference (CRISPRi), in living cells54,55. By fusing dCas9 with an activator domain, such as dCas9-VP64 and dCas9-p65, RNA-guided transcriptional upregulation of endogenous genes in mammalian cells is also achieved (Figure 4A)55–57. However, the activation efficiency of these CRISPR activators was severely low and limited its further applications. To overcome this, several second-generation CRISPR activators have been developed. A multivalent protein scaffold system termed SunTag can recruit multiple copies of protein of interest58. SunTag can be used for not only long-term imaging of single protein molecules but also potent CRISPRmediated gene activation. The SunTag-based CRISPR activator consists of dCas9 fused with ten repeats of GCN4 peptide epitopes and an anti-GCN4 antibody fused to VP64 (Figure 4B). This activator enables to recruit multiple VP64 to targeted endogenous gene for more potent activation than dCas9-VP64. dCas9-VPR was also reported as an improved CRISPR activator59. dCas9-VPR is a single polypeptide, 9 ACS Paragon Plus Environment


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dCas9 directly fused to three tandem different activator domains, VP64, p65 and Rta (Figure 4C). dCas9VPR can efficiently activate endogenous NEUROD1 in human induced pluripotent stem cells and induce its neuronal differentiation. A synergistic activation mediator (SAM) can also activate robust endogenous gene activation with a single sgRNA60 (Figure 4D). This study focused on determining optimal fusion sites of the activator domain for developing a potent dCas9 activator. Based on analysis of the crystal structure of Cas9:sgRNA:DNA complex, they found that it is possible to insert protein-interacting RNA aptamers into the stem loops of sgRNA to recruit additional activator domains. SAM consists of two chimeric proteins, dCas9 fused with VP64 and MS2-coat protein fused with p65 and HSF1 activator domains, and an sgRNA having two MS2 aptamers in stem loops termed sgRNA 2.0. MS2 aptamers in sgRNA recruits multiple p65 and HSF1 activators via the interaction between MS2 aptamers and MS2 coat proteins, and therefore this system can simultaneously recruit VP64, p65 and HSF1. It was demonstrated that SAM can efficiently activate ten different genes together in HEK293T cells. Intriguingly, because SAM can activate efficiently endogenous gene with a single sgRNA, SAM offers CRISPR-based genome-scale gene activation screening. Using SAM with lentiviral sgRNA library which can target over 20,000 human coding isoforms, they identified gain-of-function changes that confer resistance to a BRAF inhibitor in melanoma cells. The gene activation efficiency of these second generation CRISPR activators, SunTag, VPR and SAM, were directly compared in several human, mouse and fly culture cell lines61. From this report, although these activators consistently exhibited more potent gene activation compared to conventional dCas9 activators such as dCas9-VP64, the gene activation efficiency of these systems can be strikingly variable, depending on targeted gene and cell lines. In addition to transcriptional repression and activation, dCas9 is also applied to control diverse epigenetic states of targeted endogenous gene by using chimeric dCas9 fused with epigenetic modifying enzymes. These technologies are extensively summarized elsewhere62.


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Light-inducible dCas9 actuators As Cas9 was engineered into light-inducible Cas9 for spatiotemporal control, light-inducible dCas9 systems have been also developed, enabling spatiotemporal control of endogenous gene regulation. Compared to the methods for controlling the transcription of exogenous reporter gene with light63, there are few examples of light-inducible endogenous gene control. Light-inducible transcriptional effectors termed as LITEs is the first example of optogenetic endogenous gene regulation (Figure 5A)64. LITEs integrates the programmable DNA-binding TALEs and light-inducible dimerization system CRY2 and CIB148. In the dark, programmed TALE fused with CIB1 binds to targeted genomic region but CRY2 fused with VP64 diffuse freely. Blue light illumination makes CRY2 and CIB1 heterodimerized, and it induces the recruitment of VP64 into targeted genomic region, resulting in transcriptional activation of targeted gene by TALE in mammalian cells and mice. Although this LITE system has the novel capability to control endogenous gene transcription and epigenetic state at will, it requires complex and timeconsuming DNA assembly for programming TALEs to target DNA sequence. To overcome this, we developed CRISPR-Cas9-based photoactivatable transcription system (CPTS) based on dCas9 and CRY2-CIB1 light-inducible dimerization system (Figure 5B)65. CPTS can activate target endogenous gene in mammalian cells by light using corresponding sgRNAs. Furthermore, we demonstrated that it can simultaneously upregulate target endogenous genes by transfecting multiple sgRNAs. Similar system termed LACE was also developed independently by another group66.

Highly-efficient

CRISPR-Cas9-based

photoactivatable

transcription systems As the first generation CRISPR activator such as dCas9-VP64 suffered from the severely low activation efficiency, CPTS is not effective, forcing the use of multiple sgRNAs for enhancing the transcription efficiency. We have recently reported improved CRISPR-Cas9-based photoactivatable 11 ACS Paragon Plus Environment


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transcription systems which can achieve highly efficient CRISPR-mediated optical gene activation67. In this study, we applied nuclease-dead paCas9 (padCas9) to regulate the potent second-generation CRISPR activators, SunTag, VPR and SAM and found that the chimeric system based on padCas9 and SAM can induce endogenous genes more efficiently than CPTS. We further optimized this system, such as subcellular localization of components, dimerization efficiency of light-induced dimerization domains and design of activator domains, and finally established the most powerful optogenetic CRISPR activator termed Split-CPTS2.0 (Figure 5C). Using a single sgRNA, the activation efficiency of Split-CPTS2.0 is significantly higher than CPTS. For example, while CPTS with a single sgRNA targeting ASCL1 showed 22-fold light-dependent upregulation of ASCL1 mRNA in HEK293T cells, Split-CPTS2.0 can induced 1,200-fold upregulation. Split-CPTS2.0 can also efficiently activate endogenous gene in HeLa cells and primary human fetal fibroblasts. Furthermore, we demonstrated that Split-CPTS2.0 with a single NEUROD1 sgRNA can induce neuronal differentiation of human induced pluripotent stem cells via potent NEUROD1 transcriptional activation, which CPTS could not. Similar to CPTS, Split-CPTS2.0 can be activated spatially and temporally, but the switching-off kinetics of Split-CPTS2.0 is relatively slow. By extinguishing light illumination, the ASCL1 mRNA upregulated by Split-CPTS2.0 were gradually decreased but still remained for 36 hours. To achieve more temporally-confined gene activation with high efficiency, we developed another optogenetic dCas9-based transcription system, termed CPTS2.0 (Figure 5D). CPTS2.0 is not based on split dCas9 fragments fused with the Magnet system but consists of fulllength dCas9 and CRY2-CIB1 light-inducible dimerization system. CPTS2.0 is also superior to CPTS in terms of activation efficiency, but in general CPTS2.0 is less efficient than Split-CPTS2.0. In contrast to Split-CPTS2.0, the ASCL1 mRNA induced by CPTS2.0 can be decreased to baseline level within 12 h after removing light illumination, demonstrating that CPTS2.0 can be rapidly switched off by turning off blue light. Furthermore, while Split-CPTS2.0 has some background activity, we found CPTS2.0 has no significant background activity in various human cell lines. Taken together, Split-CPTS2.0 would be optimal tool when extremely potent gene activation is required, while CPTS2.0 could be an attractive option when low background and/or temporally-confined activation are required. 12 ACS Paragon Plus Environment

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Toward the improvement of optogenetic genome engineering Here we have reviewed the existing inducible Cas9 technologies, especially focusing on lightinducible Cas9. There are two types of light-inducible Cas9: one is based on chemical modification of specific amino acids or nucleotides and second on optogenetic application of genetically-encoded lightsensitive proteins consisting of twenty natural amino acids only. Although chemical modification is a straightforward approach to develop light-controlled Cas9, it could be difficult to apply these approaches into living cells and organisms because of lacking the efficient methods of protein delivery in vivo and requiring special expertise of chemistry. In contrast, “fully” genetically-encoded light-inducible Cas9 based on natural amino acids could be more easily applied to live organisms by using conventional genetic approaches. We and other groups have reported a series of genetically-encoded photoactivatable Cas9/dCas9 technologies. Intriguingly, although each of these technologies have different architectures and purposes, all of these are based on Cas9:sgRNA complex and light-sensitive proteins. Therefore, the exploitation and improvement of Cas9 toolkits and light-sensitive proteins will facilitate the further development of photoactivatable Cas9 technologies. In this section, we will look over the advances of these molecular components and discuss about the direction of improvement for advancing light-inducible Cas9 technologies.

Light-inducible protein switches Light sensitive proteins are central to optogenetics, which offers optical control of biological activity in living organisms. The most famous example is channelrhodopsin-2 (ChR2), which is a lightsensitive cation channel derived from Chlamydomonas reinhardtii. By expressing ChR2 in the brain of mouse by Cre-mediated genetic engineering, researchers can manipulate the neuronal activity in specific cell population with high temporal resolution using blue light. In addition to ChR2, many other lightsensitive proteins have been also applied to control various biological activities in vivo. In particular, 13 ACS Paragon Plus Environment


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light-inducible protein interaction systems, which can control the multimerization state of proteins by light, have been extensively exploited41. CRY2-CIB1 is one of the most popular optogenetic protein interaction systems48. These proteins were derived from Arabidopsis thaliana and the cofactor of CRY2 is flavin adenine dinucleotide which is abundant in the broad range of species, which enables to apply it in various model organisms. In the dark, CRY2 forms homodimer but not interacts with CIB1. Upon modest blue light illumination, CRY2 and CIB1 are heterodimerized. Among photoactivatable Cas9 tools, this system is applied to CPTS, CPTS2.0 and LACE. Among blue light-inducible protein interaction system, the unique feature of CRY2-CIB1 is that CRY2 itself can form oligomers in a light-dependent manner68–70. This could recruit multiple activator domains fused with CRY2 simultaneously, enhancing the gene activation efficiency. While CPTS, CPTS2.0 and LACE harnessed the oligomerization capacity of CRY2, the oligomerization of CRY2 is not necessarily appropriate in other applications. In the development of optogenetic tools based on reassociation of split enzymes, the oligomerization of dimerization domain could sterically inhibit the one-to-one interaction of split enzyme fragments. For example, we found that although split Cas9 fragments can be reassembled with rapamycin-inducible FKBP-FRB dimerization system and the Magnet light-inducible system, while CRY2-CIB1 could not provide light-induced Cas9 upregulation at all43. Furthermore, our group also developed an improved photoactivatable Cre recombinase and demonstrated that the applying Magnet system instead of CRY2-CIB1 can strikingly enhance the recombination efficiency51. These examples suggested that the oligomerization of CRY2 might negatively affect the dimerization of split enzymes, and monomeric light-inducible protein interaction systems represented by the Magnet system could be better choice for controlling split protein association with light. The dimerization efficiency and background activity of light-inducible protein interaction system are also important factors for establishing effective control of Cas9 activity with low background. In our Magnet system, we demonstrated that mutations in the residues which side-chain faces the flavin cofactor of Magnet can change not only its switching-off kinetics but also its dimerization efficiency44. Thus it


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would be interesting to improve its dimerization efficiency and/or reduce its background activity by highthroughput screening of its mutant library. The development of improved light-inducible dimerization systems would be further facilitated by growing protein engineering methodologies including directed evolution and in silico protein design71,72.Additionally, the switching on and off kinetics of light-inducible systems will be crucial parameter for rapid activation and deactivation of Cas9. Several studies show that kinetics parameters of light-sensitive domains are tunable by mutation44,73,74, which could be useful for adjusting the activation kinetics of optogenetic tools. Almost all of the existing optogenetic CRISPR-Cas9 systems are blue-light-inducible, and engineered Cas9 which can be regulated with red or far-red light remains largely elusive. Because the light having longer wavelength can penetrate more deeply living tissues and organs in vivo, red lightinducible Cas9 could manipulate genomic state in deep tissue. Also, it will also provide the possibility of combination of optogenetic Cas9 regulation with other optogenetic tools and biosensors using blue light. Recently it has been demonstrated that red light-inducible dCas9 activator based on PhyB-PIF dimerization system can upregulate reporter gene, but it remains elusive whether this system can induce endogenous genes with high efficiency75,76. Several red light-inducible dimerization systems have been developed, such as PhyB-PIF3 (or PIF6)75,77,78 and BphP1-PpsR279,80, and therefore it would be interesting to apply these in controlling Cas9 activity with red light.

Other CRISPR-associated proteins and improved Cas9s Cas9 is derived from a bacterial immunity system, and Cas9s from different species show different character. At present, SpCas9 is most popular in the world. It is not only because SpCas9 were used in the first demonstration of CRISPR-Cas9-mediated genome editing in mammalian cells, but also it has relatively higher efficiency in mammalian cells and simpler PAM (5’-NGG-3’) than other Cas9s. Furthermore, several SpCas9 mutants have been developed, which show higher specificity than the original SpCas981–84 and have different PAM sequence85,86. Because almost all of inducible Cas9s are



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based on SpCas9, these mutations improving the sequence-specificity of SpCas9 could be readily applied into these inducible Cas9. SpCas9 consists of 1368 amino acids and its large ~4.0 kb DNA sequence makes it difficult to package itself into virus vector systems. In particular, adeno-associated virus (AAV) is widely used for in vivo transduction because it is less toxic and can be efficiently transduced into various cell types. However, the packaging size of AAV is around 4.5 Kb, limiting the AAV application of Cas9. The comprehensive analysis of over 600 Cas9 orthologues sequences led to the discovery of Staphylococcus aureus Cas9 (SaCas9), which length of DNA sequence is about 3.2 Kb20. SaCas9 can efficiently induce genome editing in mammalian cells with 5’-NNGRRT-3’ PAM and the smaller size of SaCas9 enables packaging of both sgRNA under U6 promoter and CMV promoter-driven SaCas9 into AAV vector. This SaCas9 AAV can efficiently edit Pcsk9 gene in mouse liver via tail vein injection. Recently, Campylobacter jejuni Cas9 (CjCas9) was also identified, which is smaller than SaCas9 (about 3.0 Kb)87. As well as SaCas9, CjCas9 with sgRNA can be packaged into a single AAV and used for in vivo genome editing. Inducible CRISPR tools based on SpCas9 also suffer from the large DNA construct, and therefore it would be important to develop smaller inducible Cas9s for spatiotemporal control of genome in vivo. Although chemically-inducible SaCas9 has already developed88, further optimization is required because of its low efficiency. On the other hand, it might be possible to develop truncated mutant of SpCas9 and apply it into inducible Cas9. For example, based on the analysis of SpCas9 crystal structure, SpCas9 mutant which recognition lobe is partially truncated can still induce genome editing in mammalian cells89. Discovery of diverse Cas9s from different species not only provides efficient in vivo genome editing but also offers the opportunity of orthogonal endogenous gene regulation. Because each Cas9 can form Cas9:sgRNA complex with a corresponding sgRNA, different sets of Cas9:sgRNA could work independently in the same cell. SpCas9, Cas9 from Streptococcus thermophilus CRISPR1 (St1Cas9), N. meningitidis Cas9 (NmCas9) and Treponema denticola (TdCas9) are orthogonal in E. coli90. It is also demonstrated that nuclease-dead SpCas9, NmCas9 and St1Cas9 fused with transcriptional activator 16 ACS Paragon Plus Environment

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domain can orthogonally activate reporter gene in mammalian cells. Several reports demonstrated that SaCas9 is also orthogonal to SpCas976,91,92. Recently, two groups independently demonstrated inducible and orthogonal gene transcriptional regulation by combining orthogonal dCas9 activators with orthogonal chemically-inducible dimerization systems76,92. In addition, orthogonal gene regulation is also achievable by using modified sgRNAs. A scaffold RNA (scRNA), an sgRNA fused to RNA aptamer sequences which can recruit specific coat proteins can encode both target DNA sequence and regulation manner93. Using this scRNAs with the corresponding transcriptional activator and repressor fused to MS2 and PP7 coat proteins respectively, simultaneous bidirectional gene regulation was demonstrated. Because the orthogonality among scRNAs depends on the orthogonality between RNA aptamers and its binding coat protein, it does not require Cas9s from multiple species. We recently developed an optogenetic gene activation system based on RNA-protein interaction system and CRY2-CIB1 light-inducible dimerization system67, and it is interesting to investigate whether these orthogonal scRNAs can be applied orthogonal and inducible regulation of different endogenous gene. In addition to Cas9 proteins, other CRISPR-associated proteins have been identified and they showed unique features which could improve our ability in genome editing and gene regulation. Cpf1, also known as Cas12a, is a RNA-guided DNA nuclease as well as Cas9. It has been well demonstrated Cpf1 nucleases from Acidaminococcus sp. BV3L6 (AsCpf1) and Lachnospiraceae bacterium ND2006 (LbCpf1) can perform genome editing in eukaryotic cells94–96. There are several differences between Cas9 and Cpf1. While Cas9 is guided by sgRNA, fusion RNA of crRNA and tracrRNA, Cpf1 is targeted by crRNA. Cas9 orthologs have G-rich PAM but Cpf1 has a T-rich PAM. In addition, besides DNAcleavage catalytic centers, Cpf1 also has RNA-cleavage catalytic centers for processing its own crRNA array to generate mature crRNA. Processing crRNA array by Cpf1 is functional in mammalian cells, and can be used for multiplexed genome editing97. Lastly, it is suggested that Cpf1 has higher target specificity in mammalian cells than Cas998,99. On the other hand, gene regulation with nuclease-dead Cpf1 (dCpf1) has begun to be exploited. In E. coli, dCpf1 from F. novicida can repress targeted reporter



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activity, which enables comprehensive screening for determining PAM sequence100. In Arabidopsis, dCpf1 fused to three tandems of the SRDX transcriptional repressor can efficiently reduce the transcription of targeted endogenous miR159b101. It is recently demonstrated that dCpf1 fused to VPR activator domain can activate endogenous gene in mammalian cells102. Furthermore, by combining ligand-inducible dimerization domain, dCpf1-mediated endogenous gene activation has been also achieved. However, it remains elusive whether the efficiencies of these dCpf1 actuators are comparable to existing dCas9 activators. It is important to develop highly-efficient dCpf1 activators can work in mammalian cells, which could be orthogonal to existing Cas9s and expand the scope of CRISPRmediated target sequence with T-rich PAM. Also, the approaches for inducible control of Cpf1 itself is not available at present and it is also interesting to develop split Cpf1 enabling precision control of Cpf1 with external stimulus. Not only RNA-guided DNA nucleases, but also RNA-guided RNA nuclease has been recently discovered in CRISPR immune system. The first example is that Cas13a (also previously known as C2c2) from Leptotrichia shahii (LshCas13a) can be programmed to cleave specific mRNA using a single crRNA in bacteria103. Once LshCas13a:crRNA complex binds to specific mRNA, it can also work as nonspecific RNase, resulting in collateral cleavage of mRNA. Although this unique property leads to the development of highly-sensitive in vitro specific DNA and RNA detection system104, it was also main obstacle to manipulating specific RNA in living cells. For example, LshCas13a can reduce the targeted reporter mRNA level in E. coli, but also degrade non-specific mRNA, resulting in severe cell toxicity103. Despite this result, the recent study demonstrated that Cas13a from Leptotrichia wadei (LwaCas13a) can be used for RNA targeting in mammalian cells without detectable collateral RNA cleavage105. Inducible control of LwaCas13a, which would be a powerful tool for studying RNA biology, remains elusive. It would be interesting to apply the same strategy as inducible Cas9s to LwaCas13a such as usage of split architecture, inducible intein and destabilizing domain.


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Summary & Outlook Genome engineering technologies offer the targeted and specific manipulation of the genome of living organisms, and it has been significantly advanced by the emerging CRISPR-Cas9 system. CRISPRCas9 system offers a simple and efficient genome engineering and has rapidly expanded all over the world. However, there is still a lot of room for improvement. To reduce the off-target genome modification and modify targeted genome with a pinpoint accuracy, the approaches for precise control of Cas9 activity by an external stimulus have begun to be exploited. As we thoroughly reviewed above, all of inducible Cas9/dCas9 are based on engineered Cas9:sgRNA complex and ligand or light-switchable domains. Thus the expansion of CRISPR toolkits and inducible protein switches will further evolve the approaches for controlling Cas9 with high spatiotemporal precision. While various inducible Cas9/dCas9 systems have been developed, its application to the off-target problem and spatial endogenous gene regulation in vivo has not been tested extensively. Regarding offtarget genome modification, some reports show the promising examples that the specificity of Cas9mediated genome editing can be improved by tightly controlling the duration of chemically-inducible Cas9 activation29,30,32. Further characterization and optimization, such as expression level of inducible Cas9 and duration of Cas9 activation would be important to establish the versatile protocol of highlyspecific genome editing with inducible Cas9. By controlling Cas9 expression with doxycycline, temporal control of Cas9-mediated genome editing in adult mice is achieved21. However, in vivo application of inducible Cas9 which can be post-translationally controllable remains elusive and most of them have been characterized only in the context of the overexpression in culture cell lines. Then it would be essential to test whether the existing inducible Cas9 and dCas9 can be used for precise endogenous gene regulation in vivo.



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Author Information Corresponding author *Phone: +81-3-5454-6579. Fax: +81-3-5454-6579. E-mail: [email protected].

Notes The authors declare no competing financial interests.

Biographies Yuta Nihongaki, postdoctoral fellow at Johns Hopkins University (Baltimore, MD 21202), received his B.S., M.S. and Ph.D. from The University of Tokyo (Tokyo, Japan). He has worked on the development of optogenetic genome engineering technologies based on CRISPR-Cas9. His current research interests include the development of optogenetic tools and their application to understanding the molecular mechanism of signaling pathways inside primary cilia. Takahiro Otabe, postdoctoral fellow in Dr. Moritoshi Sato’s laboratory at The University of Tokyo. He received his B.S. from Nihon University, his M.S. and Ph.D. from Osaka University. His current research focuses on developing optogenetic genome engineering tools for research and therapeutic applications. Moritoshi Sato, Professor of Chemistry and Chemical Biology at The University of Tokyo (Tokyo, Japan). He received his B.S., M.S. and Ph.D. from The University of Tokyo in 1996, 1998 and 2001, respectively. He was appointed as Assistant Professor at The University of Tokyo in 2000 and promoted to Lecturer, Associate Professor and Professor in 2005, 2007 and 2017, respectively. He developed a variety of fluorescent probes to visualize molecular processes in living cells, such as second messengers and protein phosphorylation reactions. Recently, he developed photoswitching proteins, known as Magnet system, and several different tools for optogenetic manipulation of the genome, such as photoactivatable CRISPR-Cas9 system and photoactivatable Cre-loxP system. His recent works related to optogenetic technology open up new direction in life sciences.

Acknowledgement This work was supported by Japan Society for the Promotion of Science (JSPS), Japan Science and Technology Agency (JST) and Japan Agency for Medical Research and Development (AMED).


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Figure legend Figure 1. The mechanism of genome editing with programmable nucleases. (A) Schematics of genome editing. (B–D) The molecular designs of zinc finger nucleases (B), transcription activator-like effector nucleases (C) and CRISPR-Cas9 (D).

Figure 2. Architectures of genetically-encoded chemically-inducible Cas9. (A) Rapamycin-inducible split Cas9. It consists of N-terminal fragment of Cas9 fused to FRB and C-terminal fragment of Cas9 fused to FKBP. These split Cas9 fragments are inactivated in the absence of rapamycin, and rapamycin treatment induces the dimerization of FKBP and FRB, resulting in complementation of split Cas9. (B) Tamoxifeninducible intein Cas9. By inserting the 4-HT inducible intein into Cas9, the activity of the chimeric Cas9 is diminished. The inserted intein can be activated by addition of 4-HT, and it induces protein splicing, resulting in the recovery of full-length Cas9. (C) Subcellular localization control of Cas9. Cas9 fused to ERT2 is localized in cytoplasm by the interaction between ERT2 and endogenous cytoplasmic Hsp90. The small molecule 4-HT binds to ERT2 and disrupts the ERT2-Hsp90 interaction, and thus translocates the Cas9 into nucleus where targeted genome exists. (D) Conditionally-destabilized Cas9. By fusing destabilization domain to Cas9, the Cas9 is destabilized and rapidly degraded via ubiquitin-proteasome pathway. Shield-1 ligand stabilizes the Cas9 and offers inducible control of Cas9 activity. (E) Allosteric control of Cas9. In this chimeric Cas9, the REC2 domain is replaced with BCL-xL and a BH3 peptide is fused to C-terminus of the Cas9. The activity of Cas9 is turned off by interaction between BCL-xL and BH3. A3 ligand inhibits the intramolecular interaction and restores Cas9 activity.

Figure 3. Optogenetic control of CRISPR-Cas9. (A) Photoactivatable Cas9 (paCas9) consists of split Cas9 fragments fused with the light-inducible dimerization domains, Magnets. Blue light illumination induces heterodimerization of pMag and nMag, and subsequently complementation of split Cas9 27 ACS Paragon Plus Environment


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fragments, resulting in light-induced Cas9 activation. (B) Photoswitchable Cas9 (ps-Cas9) is a single component optogenetic Cas9 system. Two pdDronpa1s are inserted into the REC2 and PI domains of Cas9, respectively. These pdDronpa1s dimerizes and sterically inhibits the binding activity of Cas9. Blue light stimulation dissociates the dimerization of pdDronpa1s and thus activates ps-Cas9. (C) Cas9 fused to RsLOV2 forms a homodimer, which Cas9 activity is blocked by steric hindrance. The RsLOV2 homodimer dissociates in response to blue light, which releases the Cas9 from steric inhibition and restores its activity.

Figure 4. Representative dCas9 activators. (A) dCas9-VP64 is a first generation dCas9 activator. It is targeted to upstream sequence of transcription start site of gene of interest, and a VP64 activator domain recruits transcription complex for activating gene expression. (B) SunTag-based dCas9 activator consists of dCas9 fused with ten repeats of GCN4 epitope peptides and scFv for the GCN4 epitope combined with a VP64 activator domain. This activator can recruit 10 copies of VP64 simultaneously into targeted locus for potent gene activation. (C) dCas9-VPR is a single component activator, which employs three different activator domains, VP64, p65 and Rta. (D) SAM equips the engineered sgRNA, termed sgRNA2.0, has two MS2 RNA aptamers in its stem loops, which can be recognized by MS2 coat proteins. By using MS2 coat protein fused with p65 and HSF1 activators, SAM can recruit multiple different activators together into targeted locus by dCas9-VP64.

Figure 5. Optogenetic control of endogenous gene transcription. (A) LITE is based on programmable DNA-binding TALE domain and CRY2-CIB1 light-inducible interaction system. (B) CRISPR-Cas9based photoactivatable transcription system (CPTS) enables RNA-guided optical activation of endogenous genes, which consists of dCas9 fused to CIB1 and CRY2 fused to p65 activator domain. dCas9-CIB1 is targeted to a promoter region of target gene using sgRNAs. Blue light illumination can


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recruit p65 activator domain via CRY2-CIB1 interaction, resulting in upregulation of target endogenous gene. (C) Split-CPTS2.0 enables light-inducible recruitment of sgRNA-mediated transcriptional activators using nuclease-dead paCas9. In this system, padCas9 fused to VP64 can be targeted to gene of interest using sgRNA 2.0 having MS2 aptamers interacting with MS2 coat proteins fused to p65 and HSF1 activators. Thus Split-CPTS2.0 can accumulate multiple activator domains simultaneously in a light-dependent manner, which activates gene transcription robustly. (D) CPTS2.0 also employs MS2 RNA-protein interaction system but is based on full-length dCas9 and CRY2-CIB1. CIB1 fused to MS2 coat protein is targeted to defined locus via sgRNA-MS2 coat protein interaction. Blue light illumination induce recruitment of p65-HSF1 activators into target locus via CRY2-CIB1 dimerization for potent endogenous gene activation.



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Figure 3


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Figure 5


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For TOC only


Emerging Approaches for Spatiotemporal Control of Targeted

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