Nonviral Genome Editing Based on a Polymer-Derivatized CRISPR

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Nonviral genome editing based on a polymer-derivatized CRISPR nanocomplex for targeting bacterial pathogens and antibiotic resistance Yoo Kyung Kang, Kyu Kwon, Jea Sung Ryu, Ha Neul Lee, Chankyu Park, and Hyun Jung Chung Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00676 • Publication Date (Web): 19 Feb 2017 Downloaded from http://pubs.acs.org on February 21, 2017

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Bioconjugate Chemistry

Nonviral genome editing based on a polymer-derivatized CRISPR nanocomplex for targeting bacterial pathogens and antibiotic resistance Yoo Kyung Kang,† Kyu Kwon,‡ Jea Sung Ryu,† Ha Neul Lee,† Chankyu Park,‡ Hyun Jung Chung*,†

†

Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and

Technology, Daejeon, Republic of Korea ‡

Department of Biological Sciences, Korea Advanced Institute of Science and Technology,

Daejeon, Republic of Korea

*Corresponding author: Hyun Jung Chung, Ph.D. Email: [email protected]

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Abstract The overuse of antibiotics plays a major role in the emergence and spread of multidrugresistant bacteria. A molecularly targeted, specific treatment method for bacterial pathogens can prevent this problem by reducing the selective pressure during microbial growth. Herein, we introduce a nonviral treatment strategy delivering genome editing material for targeting antibacterial resistance. We apply the CRISPR-Cas9 system, which has been recognized as an innovative tool for highly specific and efficient genome engineering in different organisms, as the delivery cargo. We utilize polymer-derivatized Cas9, by direct covalent modification of the protein with cationic polymer, for subsequent complexation with single-guide RNA targeting antibiotic resistance. We show that nano-sized CRISPR complexes (= CrNanocomplex) were successfully formed, while maintaining the functional activity of Cas9 endonuclease to induce double-strand DNA cleavage. We also demonstrate that the CrNanocomplex designed to target mecA- the major gene involved in methicillin resistancecan be efficiently delivered into Methicillin-resistant Staphylococcus aureus (MRSA), and allow the editing of the bacterial genome with much higher efficiency compared to using native Cas9 complexes or conventional lipid-based formulations. The present study shows for the first time that a covalently modified CRISPR system allows nonviral, therapeutic genome editing, and can be potentially applied as a target specific antimicrobial.


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Introduction Over the past several decades, the overuse of antibiotics has dramatically increased the spread of multidrug-resistant bacteria.1-3 In many cases, these bacteria acquire severe virulence and infect humans which can be transmitted to other individuals, communities, healthcare units, and hospitals. The majority of these pathogens arise from the commensal bacteria in humans, which cause opportunistic infections when immunocompromised or under certain medical conditions.4 Sustained treatment with antibiotics naturally selects the mutant bacterial clones with resistance, which is acquired by changes in intrinsic expression or horizontal transfer of genes involved with enzymatic degradation or inhibition of the drug.5,6 The types of multidrug-resistant bacteria showing highest incidence rates worldwide include

methicillin-resistant

Staphylococcus

aureus

(MRSA),

carbapenem-resistant

Enterobacteriaceae (CRE), multidrug-resistant Acinetobacter baumannii (MRAB), multidrugresistant Pseudomonas aeruginosa (MRPA), vancomycin-resistant Enterococci (VRE), and more recently, vancomycin-resistant Staphlyococcus aureus (VRSA).7-10 The spread of multidrug-resistant bacteria have resulted in more limited treatment options, requiring the use or discovery of more potent drugs.11 However, the use of drugs with even higher potency would only result in the emergence of more virulent or resistant strains, and can cause higher toxicity when treated to a patient.12 Another problem is that most of the currently used drugs in the clinic for treating bacterial infections are small molecule antibiotics with broad spectrum.9 The use of drugs that can target a pathogen specifically would provide a great advantage in minimizing the selective pressure during bacterial growth. Unfortunately, the development of narrow spectrum drugs or antibody therapeutics have been disappointing due to marketing issues and technical limitations, from reasons such as the lack of specific biomarkers and acquisition of resistance.13, 14 Gene therapeutics have been introduced as an innovative approach over the conventional small molecule drugs or antibody therapeutics, due to the simplicity and versatility of designing a drug against the target with high specificity.15-18 Genetic drugs in the form of plasmid DNA, antisense oligonucleotide, small interfering RNA (siRNA), or virus-based vectors can be administered to either induce or suppress the expression of disease targets. Viral vectors are advantageous for their high transfection efficiencies, but also show limitations in the clinic due to issues on inducing cellular immune responses or antibody neutralization.16, 19, 20 In the case of mammalian cell targets, the use of siRNA to specifically -2ACS Paragon Plus Environment


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‘silence’ certain genes has been shown promising as a therapeutic and are currently under clinical trials for treating different types of cancer, glaucoma, hemophilia, and familial amyloidotic diseases.21-23 The administration of the naked drug would cause poor efficacy due to immediate enzymatic degradation in the body fluid or low delivery efficiency to the target site.22 Therefore, carrier materials such as cationic polymers, lipid-based material, inorganic nanoparticles, cell penetrating peptides, and dendrimers, have been used to condense the bioactive molecules and deliver them to the target.24-27 However, for bacterial cells, attempts to use nonviral gene delivery strategies in the clinic have been severely limited due to the poor delivery through the cell wall as well as low efficacy. Recently, the great advances in genome editing technologies have opened a new era for the development of gene therapeutics as well as genetic manipulation of model organisms.28-31 Particularly, the type II CRISPR system has provided great advantages over other conventional genome editing strategies, due to the high targeting efficiency, and simplicity in engineering.31, 32 The type II CRISPR system works by the function of the Cas9 endonuclease, which causes double-strand cleavage in the genomic DNA by assistance of a single-guide RNA (sgRNA) that binds to Cas9 and targets the genome by complementary binding.31 Most recently, there have been attempts to apply the CRISPR system as a therapeutic by inducing site-specific cleavage within the target gene.33-36 In the case of mammalian cells as targets, nonviral delivery of the Cas9 protein and sgRNA in cationic lipids,37,

38

or DNA

nanoparticle/polymer complexes39 have been reported, and introduced as a nonviral gene therapeutic. However, the methods involved noncovalent encapsulation of protein and sgRNA, which would limit their practical applications due to the low loading and packaging efficiencies, requiring administration at high dosages causing toxicity problems.40-42 In the case of bacterial cells, phagemid-based systems expressing Cas9 and sgRNA, targeting antibiotic resistance genes have been reported and shown potential use as target-specific antimicrobials.43 A nonviral delivery system for CRISPR, targeting bacterial cells, has never been reported. In this study, we introduce a nonviral delivery method for CRISPR (Cr-Nanocomplex), based on a nanocomplex of polymer-derivatized Cas9 protein and sgRNA targeting antibiotic resistance. Recombinant Cas9 endonuclease Streptococcus pyogenes (SpCas9) was covalently modified with branched polyethyleneimine, a cationic polymer, as the carrier for packaging sgRNA and enhancing their delivery into bacteria. We hypothesized that -3ACS Paragon Plus Environment

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covalently introducing a minimal amount of carrier material, onto each individual molecule of SpCas9, would allow efficient delivery into bacteria, which would be difficult using conventional noncovalent lipid-based formulations. We show that the Cr-Nanocomplex targeting mecA – the main gene involved in methicillin resistance - can be successfully delivered into Methicillin-resistant Staphylococcus aureus (MRSA) that are very difficult to transfect due to the thick bacterial cell wall. We also demonstrate that the delivered CrNanocomplex is capable of editing the bacterial genome with high efficiency, resulting in reduced growth of MRSA. The present study shows the potential applicability of the CrNanocomplex as a nonviral, target specific antimicrobial.



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Results and discussion For the efficient delivery of genome editing cargo into bacteria, the Cr-Nanocomplex system was developed utilizing polymer-derivatized Cas9 protein and complexation with sgRNA. Recombinant Cas9 endonuclease from Streptococcus pyogenes (SpCas9) was obtained by transformation of expression plasmid into E. coli competent cells and purified by affinity chromatography. The sequence of cloned SpCas9 is shown in Figure S1. The purified SpCas9 included the green fluorescent protein (GFPuv) fused with the Cas9 endonuclease for facile characterization of delivery. Figure 1a shows the SDS-PAGE results of the purified SpCas9 protein. The size of SpCas9 was shown to be ~190 kDa. The fluorescence of the SpCas9 protein was also confirmed by observing under a UV illuminator, showing a strong fluorescence signal due to the presence of GFPuv (Figure 1b). For preparing polymerderivatized Cas9 endonuclease, we attempted to modify the purified SpCas9 with branched polyethyleneimine (bPEI), via reacting the free sulfhydryl groups on the cysteine residues of the SpCas9 protein with the primary amine groups of bPEI (Figure 1c). Prior to reaction, disulfide bond prediction of the SpCas9 protein by DiANNA 1.1 showed the presence of two free sulfhydryl groups on cysteine residues 114 and 608 exposed on the surface. It has been shown in a previous study that these two cysteine residues are relatively less conserved, and their mutations to different amino acids did not affect endonuclease activity of the protein.44 Polyethyleneimine was selected for modification of the cargo, since it is one of the most widely used carrier material for gene delivery (e.g. siRNA, plasmid DNA), and is available as branched or linear type in various molecular weights.25, 27, 42, 45, 46 The branched type was chosen since it is especially highly abundant in amine functionalities, including primary amines for facile modification, and allow more efficient packaging and delivery, compared to the linear type. Direct covalent modification of the protein was chosen instead of physical encapsulation to minimize the amount of carrier material for administration, which would resolve issues on toxicity and reduced drug activity due to insufficient release. bPEI was covalently introduced to the Cas9 endonuclease to enhance bacterial delivery of the protein itself, as well as the sgRNA by inducing their packaging by electrostatic interaction. SulfoSMCC was used as the crosslinker, to first activate the amine groups of bPEI followed by subsequent reaction with SpCas9. bPEI with two different molecular weights, 2,000 Da and 25,000 Da, were used for conjugation onto SpCas9. Since modification with bPEI Mw 25,000 resulted in low yield and technical issues due to protein aggregation, SpCas9 modified -5ACS Paragon Plus Environment

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with bPEI Mw 2,000 was further characterized and utilized for bacterial genome editing. Figure 1d shows the results for the gel retardation assay to confirm the successful conjugation of bPEI onto SpCas9. It was observed that SpCas9 conjugated with bPEI (SpCas9-bPEI) appeared to migrate slightly to the (-) direction, which was opposite from native SpCas9 that showed substantial migration to the (-) direction. The results for SpCas9-bPEI can be due to the change in mobility, and possibly the clustering of protein molecules, due to modification with the polymer. Although a maximum of two bPEI molecules can be conjugated onto each protein molecule, increasing the molecular weight of the protein by only 4,000 Da, this slight change (1~2%) can still substantially affect the mobility of the protein during electrophoresis due to structural or dimensional changes.47 The theoretical charge of the GFP-fused SpCas9 protein was expected to be highly negative.37 Since bPEI is highly cationic with extremely high density of amine functionalities, their conjugation onto SpCas9 can either affect the molecular charge of the protein, or induce their clustering by electrostatic protein-polymer interactions. We also examined if crosslinking among the SpCas9 protein molecules occurred. According to SDS-PAGE, SpCas9-bPEI and native SpCas9 appeared at similar regions, showing that covalently crosslinked SpCas9 proteins were not present after the conjugation reaction (Figure S3).



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Figure 1. Preparation of polymer-derivatized SpCas9 protein. (a) Expression and purification of SpCas9 protein, characterized by SDS-PAGE. (b) Fluorescence spectometry of the purified SpCas9 showing GFP signal. Inset shows SpCas9 solution observed under a UV illluminator. (c) Synthesis of polymer-derivatized SpCas9 protein by activation of bPEI using sulfo-SMCC and reaction with the free sulfhydryl groups of SpCas9. (d) Characterization of polymer-derivatized SpCas9 by gel retardation in 0.5 % agarose.


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To prepare the Cr-Nanocomplex targeting antibiotic resistance, sgRNAs were designed to target a specific sequence within the mecA gene (Figure 2a). mecA encodes the penicillin binding protein-2a (PBP2a) and is the major antibiotic resistance gene involved in Methicillin-resistant Staphylococcus aureus (MRSA).48 Three different sgRNAs targeting different regions of mecA were designed. sgRNA(1) and sgRNA(2) were newly designed for this study, while sgRNA(3) was designed from modification of a previously reported sgRNA.43 The protospacer regions were all adjacent to a PAM sequence (NGG), from which target cleavage would occur at a site three bases upstream. sgRNAs included a CrRNA for targeting mecA, and a trans-activating RNA sequence (TracrRNA). A spacer (GG) was also included in the 5’ end to minimize off-target effects.33 Target bases for double-strand cleavage are marked in the mecA sequence, shown with the corresponding sgRNAs targeting these bases in Figure 2a. For preparation of sgRNAs, DNA templates for each sgRNA were first synthesized using primers in Figure S2 for in vitro transcription. The DNA templates for each sgRNA included a T7 promoter region, a template region for CrRNA and a template region for TracrRNA. The synthesized DNA template products are shown in Figure S4. In

vitro transcription was then performed using the synthesized DNA templates and T7 polymerase to produce each sgRNA. Figure 2b shows that three different types of sgRNAs sgRNA(1), sgRNA(2) and sgRNA(3), all targeting different regions of mecA, were successfully synthesized. All three sgRNAs were shown to have sizes of ~100 nucleotides. The functionality of the sgRNAs to guide double-strand cleavage was also examined. As the target DNA, a pure, cell-free DNA solution was prepared by RT-PCR of the 1803 bp region within the mecA gene (Figure S2) from the total RNA of cultured MRSA. sgRNA(1), sgRNA(2) and sgRNA(3) were each mixed with the purified native SpCas9 protein, and added with the PCR-amplified mecA target DNA, to induce endonuclease cleavage. Figure 2c shows that sgRNA(3) exhibited the highest cleavage efficiency, with both fragments (648 bp and 1155 bp) appearing in the gel electrophoresis results. sgRNA(2) showed a clear fragment right below the uncleaved DNA which corresponds to the 1463 bp fragment, but the other cleaved product was difficult to observe. For sgRNA(1), neither of the cleaved products were visible, showing that either the efficiency was too low for detection, or the sgRNA was non-functional in inducing specific double-strand breakage. Thus, sgRNA(3) was used for nanocomplex formation and further examination for delivery.



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Figure 2. Preparation of sgRNA targeting antibacterial resistance. (a) Design of sgRNAs targeting mecA. Three sgRNAs targeting different regions within mecA are shown by alignment. sgRNAs include a spacer (yellow), CrRNA (red) and TracrRNA (green). Target regions within mecA of MRSA include a protospacer (blue) and an adjacent motif (PAM, orange). The site for double-strand cleavage is located within the protospacer (arrowhead), between the third and fourth base (underlined) upstream from the PAM sequence. (b) Gel electrophoresis of the synthesized sgRNAs by PCR amplification of template sequences and in vitro transcription (sequences of primers shown in Figure S2). (c) Cleavage assay using PCR-synthesized mecA target DNA to examine functionality of sgRNAs with SpCas9 endonuclease. Lanes 3, 4, and 5 show products from treatment with sgRNAs (1), (2) and (3), respectively. Inset shows results from a separate experiment for the sample used in lane 5, to confirm cleavage.


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Using the synthesized sgRNA and polymer-derivatized SpCas9 protein, the CrNanocomplex were formed by adding sgRNA(3) with SpCas9-bPEI to induce the formation of self-assembled nano-sized complexes (Figure 3a). The pH during complexation was ~6.4, which is below the pKa (~8.6) of bPEI (2 kDa),49 and would protonate their amine functionalities to induce electrostatic binding with the anionic sgRNA. Dynamic light scattering measurements showed that the hydrodynamic sizes (Z-average) were 163.3 nm for the Cr-Nanocomplex, while the native complex was 82.6 nm. These results confirmed the successful formation of small, nano-sized protein-polymer conjugate/RNA complexes by charge interaction between the negatively charged sgRNA and positively charged polymer within SpCas9-bPEI (Figure 3b). Each complex would include several molecules of SpCas9bPEI and sgRNAs, forming larger complex structures, as opposed to unmodified SpCas9 which would mainly exist as a single protein bound to a single sgRNA molecule. The zeta potential values of the Cr-Nanocomplex and native complex both showed negative values, due to the presence of sgRNA bound to the surface of the protein, while that of the CrNanocomplex was less anionic (-12.1 mV) compared to the native complex (-19.0 mV). The zeta potential of SpCas9-bPEI before complexation showed a positive value (+4.0 mV), which was a significant change from that of native SpCas9 (-17.2 mV), due to the introduction of the cationic polymer. The less anionic property of the Cr-Nanocomplex would assist in enhancing their delivery into bacteria. To assess whether SpCas9 retained its endonuclease activity after polymer derivatization and subsequent complex formation, we again adopted the cleavage assay using PCR-synthesized target DNAs for mecA. Figure 3c shows the gel electrophoresis results after adding synthesized target DNA with the CrNanocomplex, to induce cleavage. One larger and one smaller DNA fragment appeared, which corresponds to the expected sizes of 1155 bp and 648 bp, showing that SpCas9, even after direct covalent modification with bPEI and complexation with sgRNA, is able to induce double-strand cleavage of the target DNA.



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Figure 3. Characterization of the Cr-Nanocomplex. (a) Schematic illustration for forming CrNanocomplex (by mixing SpCas9-bPEI with sgRNA), or native complexes of SpCas9 and sgRNA. (b) Hydrodynamic sizes of the Cr-Nanocomplex (SpCas9-bPEI/sgRNA) compared to native SpCas9/sgRNA complexes measured by dynamic light scattering. (c) Cleavage assay using PCRsynthesized mecA target DNA to examine endonuclease activity of the CRISPR nanocomplexes formed from SpCas9-bPEI and sgRNA(3).


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Polymer derivatization of the Cas9 protein was expected to enhance their uptake into bacteria compared to native Cas9. To examine bacterial uptake, SpCas9-bPEI was treated to cultured MRSA in vitro and observed by confocal microscopy. The high resolution images in Figure 4a show that SpCas9-bPEI was successfully uptaken into the bacteria, as bright green fluorescence from GFPuv can be clearly observed adjacent to the nuclear stain, while native SpCas9 did not show any significant uptake. As the control, native SpCas9 simply (noncovalently) mixed with bPEI was also examined, which also did not show any sign of uptake. The vicinity of GFP signals from SpCas9 and PI from the nuclear stain was determined by direct alignment of histograms from 2.5D images for each of the fluorescence signals, showing that only for SpCas9-bPEI, the two signals were in close vicinity. The efficiencies of uptake were also relatively quantified from low magnification images by image analysis, from which total fluorescence intensities of the GFP signals were normalized by signals from the nuclear stains (Figure 4b). To further confirm the uptake of the CrNanocomplex into bacteria, confocal image sections were also reconstructed into 3D images, showing the presence of the complexes within the bacterial cells from the overlap of fluorescence signals from the complex (SpCas9) and nuclear stain (Figure S5a-b). Histograms of the fluorescence signals were also obtained from different regions scanned within the confocal image, which showed that signals from the complex were present only at points where signals from the nuclear stain were also present (Figure S5c). Relative uptake values were shown to be 0.4273 for SpCas9-bPEI, compared to 0.0041, 0.0001, and 0.0083 for native SpCas9, native SpCas9 simply mixed with bPEI, and native SpCas9 mixed with lipofectamine, respectively. The greatly enhanced uptake of the Cas9 protein upon bPEI conjugation can be due to the highly cationic property of the polymer, or the resultant increase in polarity of the protein. Since the bPEI polymer is abundant in tertiary, secondary, and primary amine groups at high molecular densities,25 the interaction of SpCas9-bPEI to the negatively charged cell wall of Gram-positive bacteria would substantially increase compared to native SpCas9. In addition, the presence of bPEI on the surface of SpCas9 would allow the formation of clusters or condensation of the molecules. Overall, enhanced binding of SpCas9 to the bacterial cell wall would result in a higher chance of uptake, presumably by penetration through the peptidoglycan and subsequently through the cellular membrane. The strong cationic property of bPEI would also electrostatically interact with the bacterial DNA, which was expected to allow the molecular attraction of SpCas9 towards its genomic target. Using conventional lipofectamine as the carrier has been shown limitations -12ACS Paragon Plus Environment


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due to the low loading efficiency of the drug, and its difficult release into the cell.40 We anticipated that these problems could be solved by covalent modification of the SpCas9 protein with a cationic polymer, which, as long as the modification does not affect functional activity, would apply to each single molecule of protein while allowing the use of a minimal amount of carrier material. Another advantage comes from avoiding the process of encapsulation into the carrier material, which requires a release step of the cargo for delivery. Further evidence comes from treatment experiments using SpCas9 modified with bPEI Mw 25,000. Figure S6 shows that when modified with a larger bPEI polymer, the protein did not show any significant uptake when treated to bacteria. As only modification with the smaller bPEI shows high delivery efficiency, it is evident that using a small, optimal amount of carrier material is important to maximize delivery efficiency and minimize toxicity.


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Figure 4. Investigation of uptake of the polymer-derivatized SpCas9 into bacteria. (a) Confocal microscopy of MRSA treated with SpCas9-bPEI (200nM), native SpCas9 (200 nM) or native SpCas9 (200 nM) simply mixed with bPEI polymer (SpCas9+bPEI (mix)) (Green: signal from GFP fused SpCas9; red: nuclear stain with PI; scale bar 5 µm). (b) Relative uptake efficiencies determined by quantification from multiple sections of confocal images (* = p value < 0.05).



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We next investigated whether the Cr-Nanocomplex is able to edit the bacterial genome and target antibiotic resistance. The Cr-Nanocomplex formed with SpCas9-bPEI and sgRNA targeting mecA were treated in vitro to cultured MRSA, and bacterial growth was examined by subsequent culture in selective media. The cultured MRSA strains were formerly validated to have resistance to both methicillin and oxacillin. When the bacteria treated with the CrNanocomplex was cultured in suspension or plated in agar media including oxacillin (6 µg/ml), the clones with double-strand DNA breakage would not be able to grow, while the clones that were not affected would form colonies (Figure 5a). To first examine whether the Cr-Nanocomplex can induce bacterial genome editing, growth rates were determined from measuring the OD600 values, after suspension culture of the bacteria treated with the complexes (Figure 5b). It is shown that treatment with the Cr-Nanocomplex results in significant inhibition of growth, that is, a 32% decrease compared to treating SpCas9-bPEI without sgRNA as the control. To further assess the patterns of genome editing, bacteria were treated with the Cr-Nanocomplex or controls, and growth was determined by counting the number of colony forming units (CFUs) in the presence or absence of oxacillin (Figure 5c and 5e). As shown, treatment with the Cr-Nanocomplex showed a significant decrease in growth in the presence of oxacillin (65x106 CFU/ml) compared to the control (335x106 CFU/ml for SpCas9 only; 401x106 CFU/ml for SpCas9-bPEI only), while treatment with native SpCas9/sgRNA complexes showed a smaller decrease in growth (121x106 CFU/ml). Treatment with SpCas9-bPEI only, without sgRNA, did not show any significant decrease, demonstrating that reduced bacterial growth when treated with the Cr-Nanocomplex did not result from toxicity by the presence of bPEI. Surprisingly, the lipofectamine formulation of native SpCas9/sgRNA complexes showed no significant decrease in growth (361x106 CFU/ml) compared to the control of SpCas9 only. The use of lipofectamine for delivery of SpCas9/sgRNA complexes has shown substantial delivery efficiencies in mammalian cells,37,38 however showed poor delivery in the case of bacterial cells. We also took into account the fact that the culture of bacteria in presence of bioactive molecules (e.g. proteins such as Cas9) can influence growth by acting as a food source or stimulant.50 Therefore, ‘relative growth’ was calculated from 1) number of CFUs when treated with the complexes including sgRNA, normalized with 2) number of CFUs when treated with complexes excluding sgRNA. Figure 5d shows that treatment with the Cr-Nanocomplex showed a relative growth of 16.3 % compared to SpCas9-bPEI only, while the treatment with native SpCas9/sgRNA complexes resulted in a relative growth of 35.9 % compared to SpCas9 only. -15ACS Paragon Plus Environment

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The treatment with native SpCas9/sgRNA complexes in presence of lipofectamine resulted in a relative growth of 71.6%, compared to SpCas9 with lipofectamine without sgRNA. These results demonstrate that Cr-Nanocomplex formation allows sufficient delivery of the SpCas9 protein with sgRNA, and subsequent double-strand cleavage of the target DNA at a much higher efficiency compared to using native SpCas9 complexes with or without the use of the conventional lipofectamine carrier. Different concentrations of the Cr-Nanocomplex were also treated to the bacteria to determine the dose-dependent genome editing efficiencies (Figure S7). When comparing the Cr-Nanocomplex to the native complex, treatment at a lower concentration resulted in 18.7 % inhibition in relative growth, while treating at a higher concentration resulted in 57.7 % inhibition. Although treating a higher concentration resulted in a slightly higher mean value in inhibition compared to treating an intermediate concentration, the values were shown statistically significant only for the case of intermediate treatment. Treating a lower concentration of Cr-Nanocomplex would not be sufficient to exert significant genome editing efficacy, while a higher concentration might have interfered with the process of genome editing by affecting bacterial function and uptake, or stimulating bacterial growth. Another critical finding was that examining bacterial growth in the absence of oxacillin showed similar results from the values in the presence of oxacillin (Figure 5c), with 67x106 CFU/ml for the Cr-Nanocomplex, 135x106 CFU/ml for the native complex, and 414x x106 CFU/ml for SpCas9-bPEI only. Replica plating of the colonies formed from CrNanocomplex-treated bacteria was also performed, in which primary plates including oxacillin were replica plated on secondary plates without oxacillin. Results showed that all clones which formed colonies in non-selective media, also were able to grow in selective media (Figure S8). Individual clones from the plates were also subsequently cultured in suspension, and 100 % of the clones (n =20) showed substantial growth. These results show that genome editing by the Cr-Nanocomplex resulted in lethality of the bacteria, while the bacteria that tolerated the treatment continued to grow. There have been previous reports that bacterial genome editing by CRISPR can induce lethality resulting from irreparable chromosomal lesions.43,

51, 52

We can conclude that lethality also occurred for our case,

showing the potential use of the Cr-Nanocomplex for target-specific killing of multidrugresistant bacteria. In the mean time, lethality of the bacteria after genome editing gave rise to technical difficulties in their molecular characterization using standard methods as the case of mammalian cells, such as the cleavage assay or SURVEYOR. There has been a report that -16ACS Paragon Plus Environment


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Figure 5. Bacterial genome editing using the Cr-Nanoco mplex targeting mecA. (a) Schematic


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illustration showing the workflow for the genome editing experiment. (b) Growth rate of MRSA in selection media (6 µg/ml oxacillin) by suspension culture, after treating with the Cr-Nanocomplex (SpCas9-bPEI mixed with sgRNA) or SpCas9-bPEI without sgRNA (SpCas9-bPEI only). Untreated MSSA and MRSA were used as controls. (c) Examination of growth by plating and culture of the treated MRSA, in the presence (w/ oxa; 6 µg/ml) or absence (w/o oxa) of oxacillin, followed by colony counting. MRSA treated with native SpCas9 only (without sgRNA), SpCas9-bPEI only, and lipofectamine formulation of native SpCas9/sgRNA were used as controls. (d) Relative growth obtained by normalization with CFU numbers when treated without sgRNA for each sample (Relative growth (%) = [CFUcomplex x 100] / [CFUcomplex-sgRNA]; CFUcomplex = number of CFUs for bacteria treated with complex mixture; CFUcomplex-sgRNA = number of CFUs for bacteria treated with same complex mixture but only excluding sgRNA). (e) Images of colonies after treatment with the complex mixtures and culture in MRSA agar including 6 µg/ml oxacillin. (b), (c), (d) * = p value < 0.05, ** = p value < 0.01.



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plasmid-based CRISPR genome editing gave rise to escape mutants, however the target locus of these clones did not include any genomic alterations, and survival was due to dysfunction in genome editing by mutations in the delivery vector.52 The current study introduces a nonviral delivery system of CRISPR for treating antibioticresistant bacteria. We have introduced the use of polymer-derivatized Cas9 protein by direct covalent modification, which has never been reported in any of the previous studies. Compared to the previously reported noncovalent formulations,38, 39 the direct conjugation of Cas9 with polymer allows each single molecule of Cas9 protein to be bound to the carrier material, which would resolve loading efficiency issues found in the previous cases of noncovalent formulations. Also, since only one or two bPEI molecules of 2,000 Da was conjugated onto each Cas9 molecule, a minimal amount of carrier material can be used (2 wt% of protein) compared to other lipid formulations (up to 120 wt%),38 which would minimize toxicity or side effects and allow injections at higher dosages. This study also shows for the first time the application of a nonviral CRISPR system as an antimicrobial for treating multidrug-resistant bacteria. A previous study using phagemid-based transfection demonstrated that antibiotic resistance genes can be targeted and bacterial genomes can be engineered, showing the potential use of CRISPR as a target-specific antimicrobial.43 Herein, we demonstrate that nonviral, vector-free delivery of CRISPR can edit the bacterial genome and induce phenotypic changes, which would avoid immunogenicity problems and off-target effects, the major hurdles in the application of viral vectors as a therapeutic. Since bacteria have a very thick cell wall layer compared to mammalian cells, maximizing transfection efficiency would be critical, especially because most nonviral delivery systems show lower transfection efficiencies than virus-based delivery. When sufficient delivery and genome editing is achieved, this system can be applied as a target-specific or ‘narrow spectrum’ antimicrobial, which can potentially be used for treating multidrug-resistant infections in the clinic to prevent drug overuse, while controlling the further spread of antibiotic resistance.

Conclusions Our study demonstrates that the Cr-Nanocomplex formed from polymer-derivatized SpCas9 and sgRNA can be efficiently delivered and edit the genome of antibiotic-resistant bacteria. The direct covalent modification of Cas9 endonuclease with bPEI allows the formation of -19ACS Paragon Plus Environment

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small, nano-sized complexes when mixing with sgRNA. The polymer-derivatized SpCas9 shows greatly enhanced uptake into MRSA compared to native Cas9 with or without the use of conventional lipid-based formulations. We also show that the Cr-Nanocomplex is able to edit the bacterial genome of MRSA by targeting antibiotic resistance with high efficiency, showing their potential applicability as a target-specific antimicrobial. The ability for the CrNanocomplex to target different types of antibiotic resistance in various pathogenic species will have to be further investigated in the future. The possibility of utilizing the CrNanocomplex as a therapeutic in the clinic will also have to be assessed by examining efficacy in animal models. Several technical improvements are required to allow the practical applications of the Cr-Nanocomplex to replace the conventional small molecule antibiotics or viral-based delivery methods. Challenges due to bacterial overgrowth must be overcome by reducing delivery time and increasing treatment efficiency, while systemic treatment must be achieved by site-specific targeting.



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Experimental procedures Expression and purification of SpCas9 protein The SpCas9 gene from lentiCRISPR (Addgene) was cloned into pET21a (Novagen) using primers 5’-GGGCATATGGGCAGCAGCCATCACCATCATCACCACGATTACAA AGACGATGACGATAAGATGGCC-3’ and 5’-CCCAAGCTTTTTCTTTTTTGCCTGGCC GGCCTTT-3’ for SpCas9, and 5’-CCCAAGCTTATGAGTAAAGGAGAAGAAC-3’and 5’CCCAAGCTT TTATTTGTAGAGCTCATCCA-3’ for GFPuv. The SpCas9 contains 6x His, FLAG, nuclear localization sequence (NLS), SpCas9, and green fluorescent protein (GFPuv) from the N- to C-terminus. The cloned sequence was confirmed by DNA sequencing. After transforming the vector into BL21(DE3) E.coli competent cells for expression, the cells were inoculated in Luria-Bertani (LB) broth (containing 100 µg/ml ampicillin), grown at 30 °C overnight (to OD600 = ~0.4), and added with 0.5 mM isopropyl b-d-1-thiogalactopyranoside (IPTG) to induce SpCas9 expression. Cells were harvested at 16 h by centrifugation at 5,000 rpm for 10 min, and treated with lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 0.05 % β-mercaptoethanol, pH 8.0) with sonication (41% duty, pulse of 2 s and rest of 5 s for a total of 30 min on ice). The cell lysate was then incubated with Ni-NTA agarose beads (Qiagen) to bind the His-tagged SpCas9, washed, and eluted with buffer containing 100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea, 0.05% β-mercaptoethanol (pH 5.9). The eluents were then dialyzed (Spectra/Por, MWCO 50,000) against storage buffer (50 mM Tris HCl at pH 8.0, 200 mM KCl, 0.1 mM EDTA, 20% glycerol, 1 mM DTT and 0.5 mM PMSF) for a total of 12 h with buffer change every 2 h, and stored at -70 °C. The purified SpCas9 were examined by SDS-polyacrylamide gel electrophoresis. The GFP fluorescence of SpCas9 was confirmed by observing under a UV illuminator.

Design and synthesis of sgRNAs Single guide RNAs (sgRNAs) targeting the mecA gene in MRSA were designed to induce double strand breakage in the bacterial genome by SpCas9. Three different sgRNA sequences were determined according to various target regions within the mecA gene as protospacers (Figure 2a). The protospacer regions were all adjacent to a PAM sequence (NGG), from which target cleavage would occur at the site three bases upstream. sgRNAs included a CrRNA for targeting mecA, a trans-activating RNA sequence (TracrRNA). A -21ACS Paragon Plus Environment

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linker (GG) was also included at the 5’ end. Templates for the sgRNAs were generated by repeated annealing and extension of complementary oligonucleotide primers (Bioneer) with 30 cycles of annealing at 60 °C for 40 s, extension at 72 °C for 30 s using the HelixAmpTM Power-Pfu (NanoHelix), followed by gel extraction (QIAquick, Qiagen). In vitro transcription was performed using the Phage T7 RNA polymerase (Promega) in at 37 °C for 120 min. Sequences of the amplicons and primers are shown in Figure S2. The transcribed sgRNAs were purified by precipitation using 5 M ammonium acetate, followed by ethanol precipitation.

Bacterial strains and culture MRSA strains CCARM 3798, 3803, 3877 were obtained from the Culture Collection of Antimicrobial Resistant Microbes and were used as target bacteria with drug resistance. MSSA strain KCTC 3881 was obtained from the Korean Collection for Type Cultures and used as the non-resistant strain. For culture, each bacterial strains were inoculated into tryptic soy broth (TSB, BD Biosciences) and cultured in suspension at 37°C with a shaking incubator for 12-16 h. Bacterial growth and concentrations were determined by measuring the OD at 600 nm (0.4~0.6).

Preparation of polymer-derivatized SpCas9 Branched polyethyleneimine (bPEI, Mw 2,000 and 25,000) were activated by adding 16 mg of bPEI with 5 mg of sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate (sulfo-SMCC, Thermo Scientific) in ultra-pure water and reaction at 25°C for 3 h (molar ratio of bPEI; sulfo-SMCC=1:10). The reaction solutions were then dialyzed against deionized water (MWCO 500-1,000, Spectra/Por) for 48 h, and freeze-dried (FD8508, IlshinBioBase). bPEI was then reacted with the free sulfhydryl groups of SpCas9 by adding 2.1 mg of sulfo-SMCC activated bPEI with 2 mg of SpCas9 and reacted in phosphate buffer saline (PBS) at pH 6.9 for 4 h at 4 °C (molar ratio of SpCas9:bPEI = 1:100). The final product (SpCas9-bPEI) was dialyzed against storage buffer (MWCO 50,000, Spectra/Por) for 24 h at 4 °C with buffer change every 4 h, and frozen at liquid nitrogen for storage. The conjugation of bPEI onto Cas9 was characterized by gel retardation using agarose gel electrophoresis (0.5 %), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 5 %). -22ACS Paragon Plus Environment


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Formation of the Cr-Nanocomplex and characterization For the formation of Cr-Nanocomplex, SpCas9-bPEI (990 nM) and sgRNA(3) (1.8 µM) were mixed in deionized water (pH 6.5) and incubated at 25 °C for 15 min in static condition. As the control, native SpCas9 (990 nM) was mixed with sgRNA(3) (1.8 µM) in the same condition as above. For dynamic light scattering and zeta potential measurements, the complexed solutions were diluted in PBS or deionized water, to a final concentration of 168 nM SpCas9 and 300 nM sgRNA, respectively. The hydrodynamic sizes and zeta potentials of the Cr-nanocomplexes or native complexes were measured with ELSZ-2000ZS (Otsuka).

Cleavage assay for endonuclease activity To investigate whether the Cas9 protein after polymer derivatization and nanocomplex formation retained its functional activity in inducing double-strand DNA cleavage, an in vitro cleavage assay was performed using PCR-amplified template DNA derived from cultured bacteria. MRSA and MSSA strains were cultured, and the total RNAs were extracted using the Trizol reagent (Invitrogen) and reverse-transcribed using the amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT) at 60 °C for 1 min (denaturation), 25 °C for 5 min (annealing), 55 °C for 60 min (extension), and 85 °C for 1min (inactivation). cDNAs were then amplified with power pfu polymerase (Nanohelix) and specific primers for the mecA gene (Bioneer), using thermal cycling conditions: initiation at 95 °C for 2 min; 35 cycles of 95 °C for 20 s (denaturation), 59 °C for 40 s (annealing), 72 °C for 3 min 38 s (extension); and termination at 72 °C for 5 min. The amplified template DNAs were then treated with SpCas9-bPEI or native SpCas9 complexed with sgRNA(3) at a molar ratio of SpCas9:sgRNA:target DNA at 10:10:1, and incubation in Cas9 Nuclease Reaction Buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, pH 6.5) at 37 °C for 1 h. The final products were observed by agarose gel electrophoresis to confirm the presence and sizes of the DNA fragments.

Bacterial delivery of polymer-derivatized SpCas9 and confocal microscopy


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To demonstrate the delivery efficiency of polymer-derivatized SpCas9 into bacteria, confocal microscopy was performed. MRSA strains 3798 and 3803 were cultured prior to treatment as mentioned above. SpCas9-bPEI (200 nM) or native SpCas9 (200 nM) in PBS were treated to 1x107 of cultured MRSA. As the control, native SpCas9 simply mixed with bPEI was also used, by first mixing concentrated SpCas9 with bPEI (Mw 2,000), incubating for 15 min at 25 °C, and dilution (7X) in PBS for treatment (final concentration of SpCas9200 nM; bPEI- 3 µg/ml). After incubation at 37 °C for 2 h with gentle agitation using a shaking incubator, bacteria were repeatedly washed with PBS after centrifugation to remove the residual complexes. Bacteria were then fixed in 4 % paraformaldehyde solution, mounted onto microscopic slides using Vectashield (Vector Laboratories), and observed using a laser scanning confocal microscope (LSM780, Carl Zeiss). For quantification of relative uptake, bacteria were treated with the mixtures above at 4 x106/ml for 4 h. As another control, native SpCas9 was also mixed with Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol (final concentration of SpCas9 at 400 nM, 3.3x diluted reagent of Lipofectamine 3000). Low magnification images were obtained by confocal microscopy and the green fluorescence signals (SpCas9) were normalized to red fluorescence (PI stain) using ImageJ (NIH).

Evaluation of genome editing efficiency by the Cr-Nanocomplex The genome editing efficiency was evaluated by treatment of the Cr-Nanocomplex to bacteria and measuring bacterial growth in selective media. MRSA strains 3798 and 3803 were cultured prior to treatment as mentioned above. Cr-Nanocomplex was formed by mixing SpCas9-bPEI (990 nM) with sgRNA(3) (1.8 µM) and incubation at for 15 min. As the control complexes, native SpCas9 (990 nM) was mixed with sgRNA(3) (1.8 µM) in the same condition as above. Conventional lipid-based formulations were also prepared by adding the native complexes (50 µl) with Lipofectamine RNAiMAX (15.8 µl, Thermo Fisher Scientific), according to the manufacturer’s protocol. As controls for protein only (without sgRNA), SpCas9-bPEI only or native SpCas9 only were also prepared at 990 nM. All samples were diluted 6X in tryptic soy broth (final concentration of SpCas9:sgRNA = 165 nM: 300 nM), followed by treatment to 5x106 of cultured MRSA at 37 °C for 4 h with gentle agitation. The treated bacteria were washed with PBS, diluted (100X) in tryptic soy broth including 6 µg/mL oxacillin, and incubated at 37 °C for 90 min in a shaking incubator. Bacterial growth -24ACS Paragon Plus Environment


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was determined by measuring the OD at 600 nm (NanophotometerTM, Implen) after 90 min of growth. The bacteria treated with the nanocomplexes were also diluted (105X) in PBS, spread onto MRSA agar plates including 6 µg/mL oxacillin, and after incubation at 30 °C for 21 h, the colony forming units (CFUs) were counted. Replica plating was also performed by using a velvet to press the colonies from the master plate to the replica plates, and cultured at 30 °C for 12 h followed by colony counting.

Statistical analysis All statistical data were calculated and shown as mean ± standard deviation. Statistical significance was determined by obtaining the p value using the Student’s t test.

Supporting information Supplementary figures are provided as Supporting information.

Acknowledgements The study was supported by grants from the Ministry of Health and Welfare of Korea (HI14C2270 and HI15C1948 to H.J.C.) and the National Research Foundation of Korea (2015R1C1A1A02036647 to H.J.C.; 2014M3A9D9069603 and 2015R1A2A2A0106348 to C.P.). We also acknowledge Kka bi Son (KAIST), Hyunjun Kim (KAIST), Hae-Soo Kim (Yonsei University) and Sung-Min Nam (KAIST) for their help with preparation of SpCas9, bioconjugation, and image analysis, respectively.


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sgRNA

bPEI

SpCas9

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Cr-Nanocomplex

SpCas9-bPEI

MRSA

Native complex

X Bacterial DNA

Native complex

Cell wall Cytoplasm

Cr-Nanocomplex

Graphical abstract

ACS Paragon Plus Environment

Nonviral Genome Editing Based on a Polymer-Derivatized CRISPR

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