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Received 17 Oct 2016 | Accepted 6 Jan 2017 | Published 21 Feb 2017

Eunji Kim1,2,*, Taeyoung Koo1,3,*, Sung Wook Park4,5,*, Daesik Kim1,6, Kyoungmi Kim1, Hee-Yeon Cho1, Dong Woo Song2, Kyu Jun Lee2, Min Hee Jung2, Seokjoong Kim2, Jin Hyoung Kim4,5, Jeong Hun Kim4,5,7 & Jin-Soo Kim1,3,6

Several CRISPR-Cas9 orthologues have been used for genome editing. Here, we present the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9), for efcient genome editing in vivo. After determining protospacer-adjacent motif (PAM) sequences and optimizing single-guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specic, cleaving only a limited number of sites in the human or mouse genome. CjCas9, delivered via AAV, induces targeted mutations at high frequencies in mouse muscle cells or retinal pigment epithelium (RPE) cells. Furthermore, CjCas9 targeted to the Vegfa or Hif1a gene in RPE cells reduces the size of laser-induced choroidal neovascularization, suggesting that in vivo genome editing with CjCas9 is a new option for the treatment of age-related macular degeneration.

DOI: 10.1038/ncomms14500 OPEN

In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni

1 Center for Genome Engineering, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea. 2 ToolGen, Byucksan Digital Valley 6-cha, 219 Gasan Digital 1-ro, Geumcheon-gu, Seoul 08501, Republic of Korea. 3 Department of Functional Genomics, University of Science and Technology, Daejeon 34113, Republic of Korea. 4 Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, Republic of Korea. 5 FARB Laboratory, Biomedical Research Institute, Seoul National University Hospital, Seoul 03082, Republic of Korea. 6 Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea. 7 Department of Ophthalmology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Je.H.K. (email: mailto:[email protected]

Web End [email protected] ) or to J.-S.K. (email: mailto:[email protected]

Web End [email protected] ).

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14500

Genome editing has recently been democratized by the development of RNA-guided programmable nucleases repurposed from the type II clustered regularly

interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) adaptive immune system against invading genetic elements in eubacteria and archaea1. Cas9, the single effector protein component in the system, is complexed with CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) or with single-guide RNA (sgRNA) composed of essential portions of crRNA and tracrRNA to form a sequence-specic RNA-guided endonuclease (RGEN)2. A new RGEN with desired target specicity is readily prepared by replacing crRNA or sgRNA, which hybridizes with a target DNA sequence. Cas9 RGENs cleave chromosomal DNA in a targeted manner, enabling genetic modications or genome editing in cells and whole organisms38.

Cas9 derived from Streptococcus pyogenes (SpCas9), the rst Cas9 orthologue to enable targeted mutagenesis in human cells3,5,6,9, is still the most widely used among several Cas9 proteins available for genome editing. Owing to it large size (1,368 amino acids, 4.10 kbp; Fig. 1a), however, the SpCas9 gene and its sgRNA sequence cannot be packaged together into certain viral vectors such as adeno-associated virus (AAV)10 for efcient delivery into cells in vivo. Instead, the SpCas9 gene alone can be packaged into a single AAV vector in vivo11,12. Alternatively, SpCas9 can be split into two parts13,14, each can be packaged into two AAV vectors15,16. However, split SpCas9 is less active than the intact SpCas9 (refs 13,14). Furthermore, co-delivery of two AAV vectors is less efcient than the delivery of a single AAV vector in vivo.

As an alternative to SpCas9, Staphylococcus aureus Cas9 (SaCas9) can be used for genome editing17. SaCas9 is smaller (1,053 amino acids, 3.16 kbp) than SpCas9 and can be packaged, together with its sgRNA, into an AAV vector. Still, the SaCas9 gene cannot be packaged with a orescent reporter gene or two sgRNAs, essential for targeted chromosomal deletions or other rearrangements, into a single AAV vector in vivo. Furthermore, protospacer-adjacent motif (PAM) sequences recognized by SaCas9, 50-NNGRRT-30, occur less frequently than those recognized by SpCas9, 50-NGG-30, limiting targetable sites. In this regard, smaller Cas9 orthologues with different PAM sequences are highly desired to expand in vivo genome editing. Here, we present Campylobacter jejuni-derived Cas9 (CjCas9) for efcient genome editing in vitro and in vivo. CjCas9 is composed of 984 amino-acid residues (2.95 kbp) (Supplementary Fig. 1), much smaller than SpCas9 or SaCas9 (Fig. 1a), but has never been shown to induce targeted mutagenesis in human or other eukaryotic cells or organisms. In this study, we characterized PAM sequences and optimized the sgRNA length to utilize CjCas9 for genome editing in mice.

ResultsDetermination of PAM sequences recognized by CjCas9. First, we determined the PAM sequences recognized by CjCas9 in vitro (Fig. 1b). A PCR amplicon library containing a CjCas9 target sequence followed by randomized 10-base pair (bp) sequences was cleaved by CjCas9 (and SpCas9 as a control) and a sgRNA specic to the target sequence. Cleaved duplexes were subjected to deep sequencing to identify PAM sequences recognized by CjCas9. This assay revealed that CjCas9 recognized 50-NNNNACAC-30 or 50-NNNNRY

AC-30 (where R and Y stands for purines and pyrimidines, respectively) as PAMs in vitro. Using a different in vitro assay, Fonfara et al.18 reported that the optimal PAM for CjCas9 was 50-NNNNACA-30.

To conrm the PAM specicity in human cells, we performed cell-based reporter assays by co-transfecting plasmids encoding CjCas9 and its sgRNA and reporter plasmids containing the target site with variable PAM sequences between RFP and GFP sequences19 (Fig. 1c). Because the RFP sequence is fused to the GFP sequence out of frame in these reporter plasmids, cells express RFP but not GFP (RFP GFP ) in the absence

of CjCas9. When CjCas9 cleaves the target site and induces small insertions or deletions (indels), the GFP sequence can be fused to the RFP sequence in frame. These reporter assays showed that CjCas9 cleaved target sites containing 50-NNNNRYAC-30

PAM sequences in HEK 293 cells. Importantly, the cytosine nucleotide at the 30-end was essential.

Optimization of CjCas9 sgRNA length. We next optimized the sgRNA length for CjCas9-mediated genome editing in human and mouse cells (Fig. 2; Supplementary Fig. 2; Supplementary Table 1). We co-transfected CjCas9 and a series of sgRNAs with variable lengths into HEK 293 cells or NIH 3T3 cells and measured indel frequencies using targeted amplicon sequencing. The rst nucleotide in the sgRNAs was xed to an extra guanine(G) because sgRNAs are transcribed under the control of the U6 promoter, which requires a guanine at the 50 end. Unlike

SpCas9, which is most active with GX20 sgRNAs that hybridize with a 20-nucleotide target DNA sequence upstream of a PAM, CjCas9 was most active with GX22 sgRNAs that hybridize with a 22 nucleotides target sequence. GX20 and GX19 sgRNAs failed to induce indels at 8 out of 19 sites and 3 out of 4 sites, respectively in human or mouse cells.

We tested GX22 sgRNAs at 12 sites containing the optimal 50-NNNNACAC-30 PAM in human cells. All of these sgRNAs co-transfected with CjCas9 were able to induce indels at frequencies that ranged from 1.0 to 64% (215%, on average). CjCas9 also induced indels at sites with 50-NNNNGCAC-30

PAMs, 50-NNNNGTAC-30 PAMs, and 50-NNNNATAC-30 PAMs albeit less efciently (103%, 104%, and 165%, respectively).

Genome-wide target specicities of CjCas9. We then determined genome-wide specicities of CjCas9 using nuclease-digested whole-genome sequencing (WGS; Digenome-seq)2022. Cell-free genomic DNA digested with CjCas9 in vitro was subjected to WGS. Uniform cleavage patterns corresponding to on-target and off-target cleavage sites were computationally identied. CjCas9 designed to target 6 different DNA regions cleaved between one and 27 sites (74 sites, on average) in the human or mouse genome (Fig. 3ad). In parallel, we used Digenome-seq to test three SpCas9 nucleases designed to cleave sites that overlapped with CjCas9 target sites. SpCas9 cleaved 15 to 147 sites (7040 sites, on average) in the human or mouse genome, in line with our previous results showing that SpCas9 targeted to 11 different sites cleaved 9030 sites in the human genome21. One target site in the human genome contained a 50-NGGNACAC-30 PAM recognized by both

CjCas9 and SpCas9. CjCas9 and SpCas9 cleaved human genomic DNA at 5 sites and 45 sites, respectively, although the two Cas9 orthologues showed comparable indel frequencies at this particular site. Thus, the higher specicity of CjCas9 was not gained at the expense of its lower editing efciency. Strikingly, two CjCas9 nucleases targeting the Rosa26 locus or the Vegfa gene in the mouse genome cleaved genomic DNA only at the single on-target site, reminiscent of the remarkable specicity of Cpf1 nucleases22,23. Sequence logos obtained computationally by comparing in vitro cleavage sites

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14500 ARTICLE

pCMV pU6

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using Cas9 with its sgRNA

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NGS library construction

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Nucleotide at position X

Figure 1 | CjCas9 and its PAM specicity. (a) Cas9 orthologues and their sgRNAs. sgRNA structures were obtained using the mfold web server. See also Supplementary Fig. 1. (b) Schematic showing a PAM assay for characterization of PAM sequences recognized by Cas9. PCR amplicons containing a Cas9 target sequence (yellow) followed by randomized 10-bp (N10) sequences (green) were cleaved by CjCas9 in vitro. The sequence logo showing the

PAM specicity was obtained using deep-sequencing data. (c) PAM specicity in cells. Surrogate reporters encoding GFP and RFP and containing a CjCas9 target sequence with degenerate PAMs were transfected with CjCas9 and its sgRNA plasmid into HEK 293 cells. The percentage of cells that express both GFP and RFP was normalized with that obtained using the optimal reporter containing the 5-NNNNACAC-3 PAM sequence. ( ) represents a mock

control. Error bars indicate s.e.m. (n 3).

with each other unambiguously showed that these sites contained 50-NNNNACAC-30 or 50-NNNNRYAC-30 PAMs.

We chose the most promiscuous CjCas9 nuclease, which cleaved genomic DNA at 27 sites, and performed targeted amplicon sequencing to measure indel frequencies in human cells (Supplementary Table 2). Indels occurred at the on-target site but were not detectably induced at the other in vitro cleavage sites. Taken together, these results show that CjCas9 nucleases are highly specic in human cells. The remarkable specicity of CjCas9 can be

at least partially attributed to its extended, 22 nucleotides target and 4 nucleotides PAM sequences, compared to the 20 nucleotides target and 2 nucleotides PAM sequences recognized by SpCas9.

Efciency and specicity of CjCas9. We compared genome editing efciencies and specicities of CjCas9 with those of SaCas9. We chose 3 overlapping sites in the human genome with 50-NNGRRTAC-30 PAM sequences, which can be targeted by

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AAVS1-TS2

GX23 GX22 GX21 GX20 GX19 no sgRNA

0.001 0.01 0.1 1 10 100

GX23 GX22 GX21 GX20 GX19 no sgRNA

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GX23 GX22 GX21 GX20 GX19 no sgRNA

AAVS1-TS3

AAVS1-TS5

AAVS1-TS7

Tp53

GX23 GX22 GX21 GX20 no sgRNA

0.001 0.01 0.1 1 10 100

GX23 GX22 GX21 GX20 no sgRNA

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AAVS1-TS9 AAVS1-TS8 AAVS1-TS7 AAVS1-TS6 AAVS1-TS5 AAVS1-TS4 AAVS1-TS3 AAVS1-TS2 AAVS1-TS1

AAVS1-TS31 AAVS1-TS30 AAVS1-TS29 AAVS1-TS28 AAVS1-TS27 AAVS1-TS26 AAVS1-TS25 AAVS1-TS24 AAVS1-TS23 AAVS1-TS22

AAVS1-TS32

0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100

NNNNGCAC NNNNGTAC

AAVS1-TS39

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AAVS1-TS38

AAVS1-TS20

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AAVS1-TS19

NNNNATAC

AAVS1-TS36

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Indels (%)

Figure 2 | Optimization of sgRNA length for CjCas9. (a) sgRNAs with variable lengths (19 to 23 nucleotide complementary with a target DNA sequence) were designed and transfected with CjCas9 plasmid into human HEK 293 cells. Genomic DNA was isolated 48 h after transfection. Indel frequencies were analysed by targeted deep sequencing. The rst guanine nucleotide at the 5 end that does not match the target sequence is shown in lower case. PAM motifs are shown in red. Error bars indicate s.e.m. (n 3). (b) Mutation frequencies at target sites in the mouse genome. Rosa26 and Tp53-specic

gX22 guide RNAs were designed and transfected into mouse NIH 3T3 cells together with CjCas9 plasmid. Genome editing efciencies were examined by deep sequencing using genome DNA isolated from cells after 48 h of transfection. PAM motifs are shown in red. Error bars indicate s.e.m. (n 3). See also

Supplementary Fig. 2. (c) CjCas9-mediated genome editing at the human AAVS1 locus with different PAM sequences. sgRNAs targeting sites witha 5-NNNNACAC-3 PAM (green; 12 sgRNAs), 50-NNNNATAC-30 PAM (light blue; 7 sgRNAs), 50-NNNNGCAC-30 PAM (dark blue; 10 sgRNAs), and 50-NNNNGTAC-30 PAM (yellow; 8 sgRNAs) were designed and their activities examined in HEK293 cells with deep sequencing. Error bars indicate s.e.m. (n 3).

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14500 ARTICLE

AAVS1-TS1

AAVS1-TS8

19 18

CjCas9 (N=27)

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21.9

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37.7

** 61.1

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0.003 0.021 0.013 0.001 0.001

N=2 N=15

(+) SaCas9

() Nuclease (+) CjCas9 (+) SaCas9

(+) CjCas9() CjCas9 () SaCas9

Figure 3 | Genome-wide target specicities of CjCas9 nucleases examined using Digenome-seq. Human or mouse genomic DNA isolated from HeLa cells or NIH 3T3 cells (grey), respectively, was digested in vitro by Cas9 and its sgRNA targeted to the human AAVS1 locus (a,b,f) and the mouse Rosa26 (c), Vegfa (c,d) and Hif1a loci (d) and subjected to whole-genome sequencing. Circos plots show genome-wide DNA cleavage scores across the human or mouse genome. Red arrows indicate on-target sites. N indicates the number of in vitro cleavage sites identied by Digenome-seq. (a,b) Sequence logos were obtained by comparing DNA sequences at in vitro cleavage sites with each other. (b) Indel frequencies at the AAVS1-TS8 site measured using targeted deep sequencing. CjCas9 (orange) or SpCas9 (blue) targeted to the AAVS1-TS8 site was transfected into human HEK293 cells. Error bars indicate s.e.m. (n 3). (e) Indel frequencies at three AAVS1 sites targeted by CjCas9 (orange) and SaCas9 (violet).

both CjCas9 and SaCas9. CjCas9 and SaCas9 generated indels at these sites in HEK 293 cells with comparable frequencies of 357% and 3613%, respectively (Fig. 3e). We then determined genome-wide specicities of CjCas9 and SaCas9 nucleases

targeted to two of these overlapping sites using Digenome-seq (Fig. 3f). CjCas9 and SaCas9 targeted to the AAVS1-TS16 site cleaved human genomic DNA at 59 and 118 sites, respectively. CjCas9 and SaCas9 targeted to the AAVS1-TS39 site were more

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14500

ITR

NLS

polyA

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sgRNA EFS CjCas9

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sgRNA

sgRNA screening

AAV production

AAV infection

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Figure 4 | AAV-mediated mutagenesis in vitro and in vivo. (a) AAV vector encoding CjCas9 and its sgRNA. (b) Indel frequencies at the Rosa26 target site in mouse C2C12 myotubes infected with AAV-CjCas9. (c) Indel frequencies at the Rosa26 target site in TA muscles of C57BL6J mice injected with AAVCjCas9 measured at 2, 4, 8 and 32 weeks after injection. One-way ANOVA and Tukeys post hoc tests, **Po0.01, NS, not signicant. See also

Supplementary Fig. 3. (d) No off-target indels were detected at 20 homologous sites that differed from the on-target site by up to 4 nucleotides in the mouse genome. Genomic DNA isolated from AAV-CjCas9-injected TA muscles of C57BL6J mice mice at 32 weeks after injection was analysed by targeted deep sequencing. Mismatched nucleotides are shown in blue and PAM sequences in red. Red arrows indicate cleavage positions within the 20-bp target sequences. Error bars indicate s.e.m. (n 3).

specic than were those targeted to the TS16 site, cleaving human genomic DNA at 2 and 15 sites, respectively. Note that CjCas9 targeted to the TS16 site was more efcient than SaCas9 in terms of on-target indel frequency (46% versus 28%), suggesting that the higher specicity of CjCas9 was not gained at the expense of a lower on-target efciency. These results demonstrate that CjCas9 is as efcient as SaCas9 but is more specic than SaCas9 at least at the sites we tested in human cells.

All-in-one AAV vector for in vivo genome editing. With its small size, the CjCas9 gene plus a sgRNA sequence can be packaged into an all-in-one AAV vector (Fig. 4a). CjCas9 directed to the Rosa26 locus was expressed in C2C12 mouse myotubes using an AAV serotype DJ (AAVDJ) vector. Indels accumulated at the target site in a time- and dose-dependent manner with a frequency of up to 797% (Fig. 4b). Because no sites other than the on-target site were cleaved by this particular CjCas9 nuclease in the mouse genome, as shown above using Digenome-seq, we identied, using Cas-OFFinder24, potential

off-target sites that differed from the on-target site by up to 4 nucleotides in the genome. No indels were detected at the resulting 20 homologous sites by targeted deep sequencing even at day 14 post-infection (Supplementary Fig. 3; Supplementary Table 3), conrming the high specicity of this CjCas9 nuclease.

We next packaged the muscle-specic Spc512 promoter-driven CjCas9 and the U6 promoter-driven Rosa26-specic sgRNA into a muscle-tropic AAV serotype 9 (AAV9) vector25 (Fig. 4c). The resulting virus was administered via intramuscular injection into tibialis anterior (TA) muscles of C57BL6J mice (n 3 per group).

CjCas9-induced indels were observed at the target site in TA muscles with a frequency of 171% and 132%, 8 weeks and 32 weeks, respectively, after injection. No indels were detectably induced at the 20 possible off-target sites in TA muscles even 32 weeks after injection (Fig. 4d and Supplementary Table 3), although CjCas9 was still expressed (Supplementary Fig. 4). This result suggests that a long-term expression of CjCas9 via AAV in vivo does not necessarily aggravate off-target effects.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14500 ARTICLE

In vivo genome editing in the mouse retina. To show that CjCas9 can be expressed via AAV in other tissues such as retina in mice and to investigate the therapeutic potential of CjCas9-mediated gene surgery for the treatment of age-related macular degeneration (AMD), a leading cause of blindness in adults, we prepared an AAV9 vector encoding CjCas9 under the control of the elongation factor-1 short (EFS) promoter, enhanced green uorescent protein (eGFP) linked to the C terminus of CjCas9 with the self-cleaving T2A peptide, and a U6 promoter-driven sgRNA specic to the Vegfa or Hif1a gene, whose expression in the retina is associated with choroidal neovascularization (CNV)26,27 (Fig. 5a,b). We monitored the expression of CjCas9 in the eye and measured indel frequencies using targeted deep sequencing and VEGFA protein levels using ELISA, 6 weeks after the resulting viruses were administered into the eye via intravitreal injection.

In retinal pigment epithelium (RPE) cells, primary target cells for the treatment of AMD, AAV encoding the Vegfa-specic CjCas9 (AAV-CjCas9: Vegfa) achieved indels with frequencies that ranged from 22 to 30% at day 14, 28, and 42 post injection (Fig. 5c). As expected, CjCas9-linked eGFP was expressed in RPE cells (Fig. 5d and Supplementary Fig. 5). At day 42 post injection, CjCas9-induced indels were observed at Rosa26, Vegfa, and Hif1a target sites in the retina with a frequency of 4418%, 205, 5812, respectively (Fig. 5e) and in RPE cells with a frequency of 145%, 223%, and 312%, respectively (Fig. 5f). As expected, the VEGFA protein level measured using ELISA was decreased in the retina treated with AAV encoding the Vegfa- or Hif1a-specic CjCas9 (AAV-CjCas9: Vegfa and Hif1a) but not in those treated with AAV-CjCas9: Rosa26 (Fig. 5g). The VEGFA protein level was also decreased in RPE cells treated with the AAV-CjCas9: Vegfa. However, the protein level was not decreased in those with the AAV-CjCas9: Hif1a or Rosa26 (Fig. 5h), suggesting that VEGFA expression is differentially regulated in the retina and in RPE cells. We did not measure HIF-1a protein levels because HIF-1a is degraded under normoxia conditions. No off-target indels were detectably induced in these cells at one or two in vitro cleavage sites captured by Digenomeseq using the Vegfa-specic or the Hif1a specic sgRNA, respectively (Fig. 5i), ruling out the possibility that the partial suppression of VEGFA expression in the retina and RPE cells were caused by CjCas9 off-target effects.

Therapeutic genome editing for the treatment of CNV. Next, we induced CNV in the eye by laser treatment 6 weeks after injection of AAV and measured the area of CNV 1 week later (Fig. 6a). Both AAV-CjCas9: Vegfa and AAV-CjCas9: Hif1a reduced the area of CNV by 244% and 204%, respectively, compared to the AAV-uninjected negative control (Fig. 6bd). The Rosa26-specic CjCas9, used as another negative control, did not show any therapeutic effect. We noted that CjCas9 linked eGFP was expressed in RPE cells surrounding CNV, which are a major source of VEGFA in laser-induced CNV (Fig. 6b). This result shows that the reduction of CNV area coincides with targeted Vegfa mutagenesis and partial suppression of VEGFA expression in RPE cells (Fig. 5h).

We then investigated whether the partial gene knockout of Vegfa and Hif1a in RPE cells using AAV caused any side effect. It was reported that a conditional knockout of the Vegfa gene but not that of the Hif1a gene in mouse RPE cells leads to cone dysfunction28. We measured cone function using full-eld electroretinography (ERG) in laser-untreated CNV-free mice 8 weeks after injection of AAV-CjCas9: Vegfa and AAV-CjCas9: Hif1a (Fig. 6e,f). No signicant decrease in photopic response (Fig. 6e) or 30 Hz icker response (Fig. 6f) was observed in these

mice, compared to AAV-uninjected control mice. We also measured the size of the opsin-positive area, which is closely related to cone function, in contact with RPE cells expressing CjCas9. The AAV-CjCas9: Vegfa reduced the size by 3010%, compared with the AAV-uninjected control (Fig. 6g,h; Supplementary Fig. 6), suggesting that the partial Vegfa gene knockout using AAV can still cause local opsin dysfunction near Vegfa-edited RPE cells. As expected, however, the AAV-CjCas9: Rosa26 or AAV-CjCas9: Hif1a did not cause any such cone dysfunction. Taken together, these results raise a concern about targeted inactivation of Vegfa in RPE cells using AAV and suggest that Hif1a could be inactivated without causing cone dysfunction to avoid neovascularization for the treatment of AMD.

DiscussionIn this report, we have presented a small and highly specic Cas9 orthologue, derived from C. jejuni, that can be packaged with a reporter gene in an AAV vector for efcient gene surgery in vivo. The small size of CjCas9, compared with other orthologues including Neisseria meningitidis Cas9 (ref. 29), Streptococcus thermophilus Cas9 (ref. 29), SpCas9 (ref. 30) and SaCas9 (refs 17,31; Fig. 1a), allows more room for additional effectors or homology arms required for homologous recombination in AAV and other animal or plant viral vectors. Because CjCas9 is highly specic in vitro and in vivo, we expect that it will be widely used for precision genome editing in research and gene surgery in medicine.

In this study, we delivered the CjCas9 gene, its sgRNA sequence, and the GFP-coding gene to mutate three genes in two different tissues, TA muscles and eyes, in mice. CjCas9-induced indels at high frequencies in the Vegfa and Hif1a genes in vivo. HIF-1a is a hypoxia-inducible transcription factor that activates the transcription of VEGF A (ref. 32). Unlike VEGF A, a secretory protein and a primary therapeutic target for the treatment of AMD, HIF-1a has not been considered as a drug target: Indeed,

HIF1a in particular and transcription factors in general cannot be targeted directly by antibodies or aptamers or small molecules. In this study, we showed that CjCas9 targeted to the Hif1a gene in mouse eyes inactivated the gene in RPE cells efciently and reduced the area of CNV in a mouse model of AMD. Because the CjCas9 target site in the mouse Hif1a gene is perfectly conserved in the human HIF1A gene, the AAV presented in this study or its variants could be used for the treatment of human patients in the future. We expect that CjCas9 can be directed to other traditionally undruggable genes or non-coding sequences to broaden the range of therapeutic targets, making the entire human genome potentially druggable.

Methods

Animals. The care, use, and treatment of all animals in this study were in strict agreement with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and College of Veterinary Medicine and the guidelines established by the Seoul National University Institutional Animal Care and Use Committee, which granted permission to perform animal experiments. Eight-week-old, male, specic pathogen-free C57BL/6J mice (n 39) were used in this study. Mice were

maintained under a 12 h darklight cycle.

Laser-induced CNV model. After mice were anaesthetized, pupils were dilated with an eye drop containing phenylephrine (0.5%) and tropicamide (0.5%). Laser photocoagulation was performed using an indirect head set delivery system (Iridex) and laser system (Ilooda). Laser parameters were 810 nm wave length, 200 mm spot size, 800 mW power and 70 ms exposure time. Laser burn was induced three to four times around the optic disc. Only burns that produced a bubble without vitreous haemorrhage were included in the study. Seven days later, the eyes were xedin 4% paraformaldehyde for 1 h at room temperature. RPE complexes (RPE/choroid/ sclera) were prepared for immunostaining and then incubated with isolectin-B4 (Thermo Fisher Scientic, cat. no. I21413, 1:100) and anti-GFP antibody

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a b

PolyA

Exon1

TS14 ITR

U6 SgRNA EFS CjCas94736 bp

Exon2 Exon3

Exon4 Exon5 Exon6

Vegfa

Hif1a

NLS

T2A

eGFP

ITR

TS14 5

TS5

c d

Vegfa in RPE cells

NS NS

40302010 0

Indels (%)

14 28 42

Days post inj.(d)

** ***

*** ***

e g

VEGFA in retina

VEGFA in RPE cells

GFP/DAPI

Indels in retina

Indels in RPE cells

***

Rosa26 Vegfa Hif1a

0 0 0

40 20

***

100

VEGFA (pg ml)

Indels (%)

No AAV

AAV-CjCas9:

Rosa26

No AAV

AAV-CjCas9:

Vegfa

No AAV

AAV-CjCas9:

Hif1a

No AAV

AAV-CjCas9:

Rosa26

AAV-CjCas9:

Vegfa

AAV-CjCas9:

Hif1a

f h

20 10

100

***

Indels (%)

VEGFA (pg ml)

No AAV

AAV-CjCas9:

Rosa26

No AAV

AAV-CjCas9:

Vegfa

No AAV

AAV-CjCas9:

Hif1a

No AAV

AAV-CjCas9:

Rosa26

AAV-CjCas9:

Vegfa

AAV-CjCas9:

Hif1a

AAV-CjCas9: Vegfa AAV-CjCas9: Hif1a

0.05 0.11

0.1

0.01

0.05

30.72

+AAV

0.12

+AAV

AAV

21.99

0.01 0.1 1 10 100

Indels (%)

Figure 5 | In vivo genome editing with CjCas9 in the retina and retinal pigment epithelium. (a) The CjCas9 target sequences in Vegfa and Hif1a/HIF1A genes. The PAM sequence and the sgRNA target sequence are shown in red and blue, respectively. (b) All-in-one AAV vector encoding CjCas9. (c) Indel frequencies at the Vegfa target site were analysed in RPE cells using deep sequencing at day 14, 28 and 42 post-intravitreal injection of AAV-CjCas9: Vegfa. Error bars indicate s.e.m. (n 45). One-way ANOVA and Tukeys post hoc tests, NS, not signicant. (d) Representative confocal images of in vivo eGFP

expression in RPE cells of AAV-CjCas9-injected mice 6 weeks after injection (n 6). eGFP was stained with anti-GFP antibody (green). Nuclei were

counter-stained with DAPI (blue). Scale bar, 20 mm. (ei) At day 42 post injection of AAV-CjCas9, indel frequencies and Vegfa protein levels were measured in retina and RPE cells using deep sequencing and ELISA, respectively. (e,f) Indel frequencies at the Rosa26, Vegfa and Hif1a target sites in the retina (e) and RPE cells (f). Error bars indicate s.e.m. (n 4 for AAV-uninjected control, n 5 for AAV-CjCas9). Students t-tests, *Po0.05, ***Po0.001.

(g,h) VEGFA levels measured by ELISA in the retina (g) and RPE cells (h), respectively. Error bars indicate s.e.m. (n 67). One-way ANOVA and Tukeys

post hoc tests, *Po0.05, ***Po0.001. (i) Indel frequencies at in vitro cleavage sites identied by Digenome-seq. Genomic DNA isolated from RPE cells treated with AAV-CjCas9 at 6 weeks post injection was subjected to targeted deep sequencing. Mismatched nucleotides are shown in blue and

PAM sequences in red. Red arrows indicate cleavage positions within the 22-bp target sequences.

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(Abcam, ab6556, 1:100) overnight at 4 C. The RPE complex was at-mounted and viewed with a uorescent microscope (Eclipse 90i, Nikon) or a confocal microscope (LSM 710, Carl Zeiss) at a magnication of 100. The CNV area was measured

using Image J software (1.47v, NIH) by blinded observers. An average of 34 CNV areas per eye were analysed. Each group consisted of 1718 eyes.

Construction of cjCas9 and sgRNA plasmids. A human codon-optimized CjCas9-coding sequence, derived from Campylobacter jejuni subsp. Jejuni NCTC 11168, was synthesized with a nuclear localization signal and an HA epitope at its C-terminal end (GeneArt Gene Synthesis, Thermo Fisher Scientic) and cloned into the p3s plasmid4. The trans-activating crRNA (tracrRNA) sequence and the

CNV

(Day 0) Intravitreal injection of AAV-CjCas9

(Day 42) Laser-induced CNV

(Day 49) CNV analysis in RPE flat-mount

Merge

No AAV

IB4 GFP GFP-enlarged

AAV-CjCas9: Rosa26 AAV-CjCas9: Vegfa AAV-CjCas9: Hif1a

d e f

Laser induced CNV area Photopic response 30Hz flicker response

150 * ****

***

Relative CNV area (%)

100

0 No AAV

Amplitude (V)

150

100

0 0

Amplitude (V)

AAV-CjCas9:

Rosa26

AAV-CjCas9:

Vegfa

AAV-CjCas9:

Hif1a

No AAV

AAV-CjCas9:

Vegfa

AAV-CjCas9:

Hif1a

No AAV

AAV-CjCas9:

Vegfa

AAV-CjCas9:

Hif1a

No AAV

ONL

AAV-CjCas9: Rosa26 AAV-CjCas9: Vegfa AAV-CjCas9: Hif1a

Opsin/DAPI

NS * * *

Relative opsin area (%)

150

100

0 No AAV

AAV-CjCas9:

Rosa26

AAV-CjCas9:

Vegfa

AAV-CjCas9:

Hif1a

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gene with a nuclear localization signal and an HA tag at the C terminus were constructed. sgRNA transcription was driven by the U6 promoter and CjCas9 expression was controlled by the EFS promoter in C2C12 myoblast cells or by the Spc512 promoter in TA muscles of C57BL6J mice mice. For retinal delivery, an AAV vector encoding CjCas9 under the control of the EFS promoter, enhanced green uorescent protein (eGFP) linked to the C terminus of CjCas9 with the self-cleaving T2A peptide, and a U6 promoter-driven sgRNA specic to the Vegfa or Hif1a gene was constructed.

Production and characterization of AAV vectors. To produce AAV vectors, they were pseudotyped in AAVDJ or AAV9 capsids. HEK293T cells were transfected with pAAV-ITR-CjCas9-sgRNA, pAAVED2/9 and helper plasmid.HEK293T cells were cultured in DMEM with 2% FBS. Recombinant pseudotyped AAV vector stocks were generated using PEI coprecipitation with PEIpro (Polyplus-transfection) and triple-transfection with plasmids at a molar ratio of 1:1:1 in HEK293T cells. After 72 h of incubation, cells were lysed and particles were puried by iodixanol (Sigma-Aldrich) step-gradient ultracentrifugation. The number of vector genomes was determined by quantitative PCR.

AAV transduction in mouse myoblast cells. Mouse myoblast cells were infected with AAVDJ-CjCas9 at different viral doses (multiplicity of infection (MOI): 1, 5, 10, 50, and 100 determined by quantitative PCR) and maintainedin DMEM with 2% FBS. At different time points, cells were collected for targeted deep sequencing. An MOI of 1 was estimated with one infectious virus particle in 100 total viral particles determined by quantitative PCR.

Intramuscular injection of AAV. AAV was administered to 8-week-old young adult male C57BL/6J mice anaesthetized with 24% isourane. The mice were injected intramuscularly with AAV9-CjCas9 (1 1011 viral genome) in physiolo

gical saline (40 ml) using an ultra-ne insulin syringe with a 31 G needle (BD).

As a negative control, C57BL/6J mice were injected with physiological saline (40 ml) only.

Intravitreal injection of AAV. 8-week-old mice were anaesthetized with an intraperitoneal injection of a mixture of tiletamine and zolazepam (1:1, 2.25 mg per kg body weight) and xylazine hydrochloride (0.7 mg per kg body weight).AAV9-CjCas9 (2 1010 viral genome in 2 ml) was intravitreally injected using

a Nanol syringe with a 33 G blunt needle (World Precision Instruments Inc.) under an operating microscope (Leica Microsystems Ltd.).

Immunouorescent staining and imaging of retinal tissue. For the analysis of opsin-positive area, formalin-xed parafn-embedded samples were prepared at day 42 post injection (n 4). Cross-section samples were immunostained with

anti-HA antibody (Roche, 3F10, 1:1,000), anti-opsin antibody (Millipore, AB5405, 1:1,000), and Alexa Fluor 488 or 594 antibodies (Thermo Fisher Scientic, 1:500).The opsin-positive area corresponding to RPE cells expressing HA-tagged CjCas9 was measured using Image J software (1.47v, NIH) by blinded observers. For the distribution of CjCas9 and eGFP, the eyes were xed in 4% paraformaldehyde for 1 h at room temperature. RPE complexes (RPE/choroid/sclera) were prepared for immunostaining and then incubated with anti-GFP antibody (Abcam, ab6556, 1:100) overnight at 4 C. After stain with Alexa Fluor 488 antibodies (1:500), the RPE at-mounts was imaged using a confocal microscope (LSM 710, Carl Zeiss).The scanning parameters were as follows: scaling (x 0.042 mm per pixel,

y 0.042 mm per pixel, z 0.603 mm per pixel), dimensions (x 1,024, y 1,024,

channels: 2, 8-bit) with objective C-Apochromat 40 per 1.20 W Korr M27.

ZEN 2 software was used to process the images.

Figure 6 | CjCas9 targeted to Vegfa or Hif1a reduces the area of laser-induced CNV in mice. (a) At day 42 post injection of AAV-CjCas9, mice was treated with laser to induce choroidal neovascularization (CNV). One week after laser treatment, the CNV area was analysed. (b) In vivo expression of eGFP coexpressed with CjCas9 in laser-induced CNV. Representative confocal images of eGFP expression in the RPE of laser-induced CNV, 6 weeks after injection of AAV-CjCas9: Hif1a. eGFP was stained with anti-GFP antibody (green), CNV was stained with anti-IB4 antibody (red), and nuclei were counter-stained with DAPI (blue). Scale bar, 200 mm. Arrows indicate eGFP-expressing RPE cells. Scale bar, 100 mm (enlarged image). (c) Representative laser-induced CNV stained with isolectin B4 in the mouse eye injected with AAV-CjCas9 targeted to Rosa26, Vegfa or Hif1a. Scale bar, 200 mm. (d) The CNV area.

Error bars indicate s.e.m. (n 1718). One-way ANOVA and Tukeys post hoc tests, *Po0.05; **Po0.01; ***Po0.001; NS, not signicant. (e,f) At day 56

post AAV injection, full-eld electroretinogram (ERG) was performed to evaluate cone function in mice. There was no signicant decrease of b-wave of photopic response (e) and 30 Hz icker response (f) in both AAV-CjCas9: Vegfa (n 8) or AAV-CjCas9: Hif1a (n 6) treated mice, compared to normal

control mice (n 8). Error bars indicate s.e.m. (n 68). (g,h) Opsin-positive areas in the retina at day 42 post injection. (g) Representative images of

opsin-positive areas in contact with RPE cells expressing HA-tagged CjCas9 in AAV-CjCas9: Rosa26, Vegfa or Hif1a injected mice compared with the AAV-uninjected negative control mice (no AAV). Opsin (red) and DAPI (blue). Scale bar, 20 mm. ONL, outer nuclear layer; IS, inner segment of photoreceptor cells; OS, outer segment of photoreceptor cells. See also Supplementary Fig. 6. (h) Relative opsin areas of the AAV-CjCas9-injected mice were normalized to that of the AAV-uninjected negative control mice. Error bars indicate s.e.m. (n 4). One-way ANOVA and Tukeys post hoc tests,

*Po0.05.

precursor CRISPR RNA (pre-crRNA) sequence were fused with a GAAA or TGAA linker to form a sgRNA sequence (Supplementary Fig. 1). sgRNAs were transcribed under the control of the U6 promoter. We used addgene plasmid (# 61591) for SaCas9 expression.

In vitro PAM identication. The recombinant Cas9 protein was expressed inE. coli and puried as described previously3. sgRNAs were transcribed usingT7 RNA polymerase as described3. To make a randomized PAM (N10) library, the

DNA sequence including the AAVS1-TS1 target site cloned in a plasmid was amplied with a randomized primer. After gel purication, the amplicon library (1 mg) was digested with the SpCas9 or CjCas9 protein and in vitro transcribed sgRNA for 30 min at 37 C. Digested library was puried by column ltration and subjected to deep sequencing using Miseq (Illumina). Miseq reads that perfectly matched the reference sequence were sorted. The randomized PAM region was extracted and analysed with WebLogo.

PAM characterization using cell-based reporter assays. The AAVS1-TS1 target sites with variable PAM sequences, which were randomized at position X (50-NNNNXCAC-30, 50-NNNNAXAC-30, 50-NNNNACXC-30 and 50-NNNN

ACAX-30), were synthesized (Macrogen, Inc.) and cloned in a surrogate reporter plasmid encoding RFP and GFP19. To determine optimal PAM sequences, each of the resulting reporter plasmids (100 ng) and plasmids encoding CjCas9 and its sgRNA (225 and 675 ng, respectively) were co-transfected into HEK293 cells(1 105) using lipofectamine 2000 (Invitrogen). At day 2 post-transfection, the

fraction of GFP and RFP double-positive cells was determined by ow cytometry (BD Accuri C6, BD).

Cell culture and mutation analysis. HEK 293 (ATCC, CRL-1573) cells and mouse NIH 3T3 (ATCC, CRL-1658) cells were maintained in DMEM supplemented with 100 units per ml penicillin, 100 mg ml 1 streptomycin, and 10% fetal bovine serum (FBS). sgRNA plasmid (750 ng) and CjCas9 plasmid (250 ng) were transfected into cells (0.5B1 105) with lipofectamine 2000 (Invitrogen). After

48 h of transfection, genomic DNA was isolated using a DNeasy Blood & Tissue kit (Qiagen) and on-target or off-target loci were amplied using specic primers (Supplementary Table 4) for targeted deep sequencing. Deep-sequencing libraries were generated by PCR. TruSeq HT Dual Index primers were used to label each sample. Pooled libraries were subjected to paired-end sequencing (LAS, Inc.). Indel frequencies were calculated as described previously20.

Digenome sequencing. Digenome-seq was performed as described previously20,21. Genomic DNA was isolated using a DNeasy Tissue kit (Qiagen) according to the manufacturers instructions. Genomic DNA (8 mg) with CjCas9 or

SaCas9 protein (300 nM) and sgRNA (900 nM) in a 400 ml reaction volume(100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, and 100 mg ml 1 BSA) and incubated the mixture for 8 h at 37 C. Digested genomic DNA was incubated with RNase A (50 mg ml 1) for 30 min at 37 C and puried again with a DNeasy Tissue kit (Qiagen). Digested DNA was fragmented using the Covaris system and ligated with adaptors for library formation. DNA libraries were subjected to WGS using an Illumina HiSeq X Ten Sequencer at Macrogen. We used the Isaac aligner to generate a Bam le using the following parameters: ver. 01.14.03.12; Human genome reference, hg19 from UCSC (original GRCh37 from NCBI, Feb. 2009), Mouse genome reference, mm10 from UCSC; Base quality cutoff, 15; Keep duplicate reads, yes; Variable read length support, yes; Realign gaps, no; and Adaptor clipping, yes (adaptor: 50-AGATCGGAAGAGC*-30, 50-*GCTCTT

CCGATCT-30)33.

AAV vectors encoding CjCas9 and its sgRNA sequences. AAV inverted terminal repeat-based vector plasmids carrying a sgRNA sequence and the CjCas9

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Genomic DNA extraction. For DNA extraction from muscle, muscle tissue was homogenized using tungsten carbide beads (3 mm; Qiagen) and a TissueLyser II (Qiagen). For extraction from RPE, after imaging of RPE at-mounts, tissue samples were washed in PBS. RPE cells were mechanically isolated from choroid/ sclera by vortexing for 30 s in lysis buffer (NucleoSpin Tissue, Macherey-Nagel), as described34. Genomic DNA from the remnant choroid/sclera tissues was analysed to conrm complete isolation of RPE cells. Genomic DNA was analysed by targeted deep sequencing.

Mouse Vegfa ELISA. At day 42 post injection, whole RPE complexes were separated from neural retina tissue and frozen for further analysis. Sample tissues were lysed with Cell Lysis Buffer (120 ml) (CST #9803) and Vegfa protein levels were measured using a mouse VEGF Quantikine ELISA kit (MMV00, R&D systems) according to the manufacturers instructions.

ERG analysis. Mice were dark-adapted over 16 h. Mice were anaesthetized with an intraperitoneal injection of a mixture of tiletamine and zolazepam (1:1, 2.25 mg per kg body weight) and xylazine hydrochloride (0.7 mg per kg body weight). Pupils were dilated with an eye drop containing phenylephrine (0.5%) and tropicamide(0.5%). Contact lens electrodes were placed on both eyes with a drop of methylcellose. Full-eld ERGs were recorded as described35 by using the universal testing and electrophysiologic system 2000 (UTAS E-2000, LKC Technologies, Gaithersburg, MD). The responses were recorded at a gain of 2 k using a notch lter at 60 Hz, and were bandpass ltered between 0.1 and 1,500 Hz. In the light-adapted state (photopic), with a 30 cd/m2 background light to desensitize the rods and isolate cones, cone responses were recorded in response to single ashed of 0 dB for photopic response, and a icker sequence of 30 Hz, averaging 20 responses. The amplitude of the a-wave was measured from the baseline to the lowest negative-going voltage, whereas peak b-wave amplitudes were measured from the trough of the a-wave to the highest peak of the positive b-wave.

Western blotting. The CjCas9 protein expressed in TA muscles of C57BL/6J mice at 8 months after injection of AAV was detected using western blotting. Samples containing equal amounts of protein (20 mg) were analysed; Cas9 and GAPDH were detected with an anti-HA high afnity antibody (Abcam, ab9110, 1:5,000) and an anti-GAPDH antibody (Abcam, ab9485, 1:2,500), respectively. Goat anti-rabbit IgG-HRP antibody (Abcam, ab6721, 1:5,000) was used for signal detection. ImageQuant LAS4000 (GE healthcare) was used for digital imaging. Uncropped scans of blots in main gures are presented in Supplementary Fig. 4.

Statistical analysis. No statistical methods were used to predetermine sample size for in vitro or in vivo experiments. All group results are expressed as means.e.m., if not stated otherwise. Comparisons between groups were made using the two-tailed Students t-test or one-way analysis of variance (ANOVA) and Tukeys post hoc tests for multiple groups. Statistical signicance as compared with untreated controls was denoted with *Po0.05, **Po0.01, ***Po0.001 in the gures and gure legends. Statistical analysis was performed in Graph Pad

PRISM 5.

Data availability. The deep-sequencing data from this study have been submitted to the NCBI Sequence Read Archive under accession number SRP095501 and SRP095507. The data that support the ndings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by IBS-R021-D1 (to J.-S.K.), the Korea Health technology

R&D Project through the Korea Health Industry Development Institute (Grant Number:

HI16C0426 to S.K.), the Pioneer Research Program of NRF/MEST (2012-0009544 to

Je.H.K.), the Bio & Medical Technology Development Program of the National Research

Foundation and MSIP (NRF-2015M3A9E6028949 to Je.H.K.). We thank Kwang-eun

Kim for experimental support.

Author contributions

J.-S.K. and J.H.K. supervised the research. All the other authors performed the

experiments.

NATURE COMMUNICATIONS | 8:14500 | DOI: 10.1038/ncomms14500 | http://www.nature.com/naturecommunications

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14500

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Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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Web End =naturecommunications Competing nancial interests: J.-S.K. is a co-founder and shareholder of ToolGen, Inc.

D.W.S., K.J.L., M.H.J. and S.K. are employed by ToolGen, Inc. Je.H.K., S.W.P., D.W.S.,

E.K., and S.K. have led patent applications based on this work. The remaining authors

declare no competing nancial interests.

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How to cite this article: Kim, E. et al. In vivo genome editing with a small

Cas9 orthologue derived from Campylobacter jejuni. Nat. Commun. 8, 14500

doi: 10.1038/ncomms14500 (2017).

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12 NATURE COMMUNICATIONS | 8:14500 | DOI: 10.1038/ncomms14500 | http://www.nature.com/naturecommunications

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Abstract

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Several CRISPR-Cas9 orthologues have been used for genome editing. Here, we present the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9), for efficient genome editing in vivo. After determining protospacer-adjacent motif (PAM) sequences and optimizing single-guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specific, cleaving only a limited number of sites in the human or mouse genome. CjCas9, delivered via AAV, induces targeted mutations at high frequencies in mouse muscle cells or retinal pigment epithelium (RPE) cells. Furthermore, CjCas9 targeted to the Vegfa or Hif1a gene in RPE cells reduces the size of laser-induced choroidal neovascularization, suggesting that in vivo genome editing with CjCas9 is a new option for the treatment of age-related macular degeneration.

Details

Title

In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni

Author

Kim, Eunji; Koo, Taeyoung; Park, Sung Wook; Kim, Daesik; Kim, Kyoungmi; Cho, Hee-yeon; Song, Dong Woo; Lee, Kyu Jun; Jung, Min Hee; Kim, Seokjoong; Kim, Jin Hyoung; Kim, Jeong Hun; Kim, Jin-soo

Pages

14500

Publication year

2017

Publication date

Feb 2017

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms14500

ProQuest document ID

1870238343

In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni

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