Targeted mutagenesis using CRISPR/Cas system in medaka

1Genome editing with artificial nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has become a powerful tool for approaches involving reverse genetics in a wide range of organisms. These enzymes efficiently induce site-specific DNA double-strand breaks (DSBs), resulting in targeted gene disruptions by insertions and deletions (indels) or targeted gene integrations by homologous recombination. However, since the DNA-binding domain of these nucleases determines their site specificity, re-engineering the binding domain sequence is essential for each new target site.

2The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has recently emerged as an RNA-guided endonuclease (RGEN) for targeted genome editing. CRISPR and Cas proteins are essential components of the adaptive immune system in bacteria and archaea to detect and silence invading viruses and plasmids. In type II CRISPR system, Cas9 protein, CRISPR RNAs (crRNAs), and trans-activating crRNA (tracrRNA) form ribonucleoprotein complexes that induce site-specific DNA cleavage guided by crRNAs. The recognition specificity of Cas9 endonuclease from Streptococcus pyogenes type II CRISPR/Cas system can be programmed only by a synthetic single-guide RNA (sgRNA) consisting of a fusion of crRNA and tracrRNA. Recent studies have shown that Cas9 and engineered sgRNA are the only components necessary and sufficient for targeted DNA cleavage and efficient genome editing in cultured human cells, mice, Drosophila, Caenorhabditis elegans, and zebrafish. Because of its simple customizing process compared to the assembling of TALEN or ZFN modules, the CRISPR/Cas-mediated RGENs have the potential for being developed as a robust and efficient tool for genome editing.

3However, RGENs still pose several unanswered questions for research applications. It remains unclear whether the selection of the target sequence is crucial to achieve effective targeted cleavage. Site specificity of DNA cleavage by the Cas9 endonuclease from S. pyogenes depends on two factors, one being the base-pair complementarity between the first 20 nucleotides (nts) of a guide RNA and a target DNA sequence, and the other being the sequence “NGG”, referred to as the protospacer adjacent motif (PAM), adjacent to the complementary region in the target site. Hwang et al. described that sgRNAs transcribed by a T7 RNA polymerase require their target sequence in the form 5′-GG-N18-NGG-3′ because GG is added at the 5′ ends of the transcripts initiated at the T7 promoter. On the other hand, a study in cultured human cells showed that double mismatches at the 5′ ends of the sgRNAs are tolerated, and in vitro studies showed that the GG required by the T7 promoter do not affect cleaving activities. Furthermore, Ran et al. showed that 5′ extension of sgRNA sequences could not contribute to Cas9 targeting specificity in cultured human cells because the 5′ extensions were processed. In fact, Hwang et al. also reported that sgRNAs transcribed by T7 polymerase could target the genomic sequences without the 5′ ends GG in zebrafish. However, there are very few in vivo studies that investigate the effects of the 5′-end sequences on the cleaving activities. Second, and more importantly, the relatively short target sequence of 20 bp or fewer for the RGENs raises questions about their specificity. It was reported that potential off-target sites including both a PAM and a perfect match of at least 12 bp at the 3′ end of the 20-bp targeting sequence were not disrupted in mice, Drosophila, and C. elegans. However, off-target sites harboring up to five mismatches were mutagenized in cultured human cells and zebrafish. Additionally, a more recent work indicates that the off-target sites that have up to 3 bp mismatches except in the 8-bp sequence adjacent to a PAM can be disrupted by RGENs. These suggest that the RGENs can also induce off-target alterations in other organisms.

4Here, we showed successful targeted mutagenesis using CRISPR/Cas-mediated RGENs in medaka (Oryzias latipes), which is a small freshwater teleost and is used as a vertebrate model in a wide range of scientific studies. We injected fertilized eggs with capped RNAs encoding the S. pyogenes Cas9 nuclease and sgRNAs transcribed by the T7 promoter and introduced somatic and germ line mutations in their target sites with frequencies comparable to TALEN-mediated mutagenesis studied previously by us. We showed that the RGENs introduced mutations at the target genomic sequence without GG at the 5′ end as efficiently as those starting with GG. We also revealed that the off-target alterations in the RGEN-injected fish were introduced at genomic loci harboring double mismatches in the 18-bp sequence located at the 3′ ends of the targeting sequence. Additionally, we exhibited that these off-target effects were reduced by lower dosages of sgRNA.

5A d-rR strain was used in this study. Fish were maintained in an aquarium with recirculating water in 14/10-h day/night cycle at 26°C. The care and use of animals in this study were in accordance with the guidelines of the Animal Experimentation Committee of Kyoto University.

6A Cas9 expression vector for SP6 in vitro transcription, pCS2+hSpCas9, was generated in this study. DNA sequence encoding the human codon-optimized S. pyogenes Cas9 nuclease was amplified from the pX330 (Addgene Plasmid 42230) by PCR using the primers: hSpCas9FW and hSpCas9RV (supplementary material Table S1). The resulting PCR product was cloned into the BamHI/XbaI site of pCS2+MT vector. This expression vector will be made available via Addgene (http://www.addgene.org).

7The pDR274 vector (Addgene Plasmid 42250), harboring a T7 promoter positioned upstream of a partial guide RNA sequence was used for sgRNA expression. Appropriately designed oligonucleotides were synthesized with oligonucleotide purification cartridge (OPC) purification at Operon Biotechnologies (Tokyo. Japan). A pair of oligonucleotides (final concentration: 10 µM each) was annealed in 10 µL of annealing buffer (40 mM Tris-HCl [pH 8.0], 20 mM MgCl2, and 50 mM NaCl) by heating to 95°C for 2 min and then cooling the mixture slowly to 25°C in 1 h. The pDR274 vector was digested with BsaI-HF (New England Biolabs), and the annealed oligonucleotides were ligated into the pDR274 vector. Sequences of the genomic target sites and the annealed oligonucleotides are listed in supplementary material Table S2.

8The Cas9 expression vector was linearized by NotI digestion. Capped RNA was synthesized using the mMessage mMachine SP6 Kit (Life Technologies), and then purified using the RNeasy Mini Kit (Qiagen). The sgRNA expression vectors were digested by DraI, and the sgRNAs were synthesized using the AmpliScribe T7-Flash Transcription Kit (Epicentre). The synthesized sgRNAs were purified by ammonium acetate precipitation.

9These RNA sequences were diluted to appropriate concentrations and injected approximately 2–4 nL of the RNA mixture into fertilized eggs before the first cleavage, as described previously.

10Embryos were lysed individually at 3 days post fertilization (dpf) in 25 µL of alkaline lysis buffer containing 25 mM NaOH and 0.2 mM EDTA (pH 8.0) and incubated at 95°C for 15 min after breaking the egg envelope (chorion) with forceps. Samples were neutralized with 25 µL of 40 mM Tris-HCl (pH 8.0) and used as genomic DNA samples.

11Heteroduplex mobility assay (HMA) was performed to detect RGEN-induced mutations. A 146-bp fragment containing the entire genomic target sequence of the DJ-1 gene was amplified using primers DJ1-FW2 and DJ1-RV2 (supplementary material Table S1). The reaction mixture contained 1 µL of genomic DNA as template, 1× PCR buffer for KOD FX, 0.4 mM of each dNTP, 0.2 µM of each primer, and 0.05 unit of KOD FX (Toyobo) in a total volume of 10 µL. The cycling conditions were as follows: one cycle at 94°C for 2 min, followed by 35 cycles of 98°C for 10 sec, 56°C for 20 sec, and 68°C for 20 sec. The resulting amplicons were analyzed using a microchip electrophoresis system (MCE-202 MultiNA; Shimazu) with the DNA-500 reagent kit.

12For sequence analysis at DJ-1 locus, the genomic region including the target site of sgRNAs was amplified with KOD -plus- Neo DNA polymerase (TOYOBO) using the primers DJ1-FW3 and DJ1-RV3 (supplementary material Table S1). The PCR conditions were as follows: one cycle at 94°C for 2 min, followed by 35 cycles of 98°C for 10 sec, 58°C for 30 sec, and 68°C for 30 sec. The PCR amplicons were subcloned into the EcoRI/XhoI site of the pBluescript KS II (+) vector. The fragment containing the cloned genomic sequence was amplified from each colony using the M13 forward and reverse primers (supplementary material Table S1). Each fragment was sequenced using a T7 promoter primer (supplementary material Table S1).

13A 285-bp fragment including the genomic target sequence of the sgRNA was amplified using the primers DJ1-FW2 and DJ1-RV1 (supplementary material Table S1). The reaction mixture contained 2 µL of genomic DNA template, 1× reaction buffer, 0.8 mM of each dNTP, 1.5 mM of MgCl2, 0.2 mM of each primer, and 0.5 unit of HybriPol DNA Polymerase (Bioline, London) in a total volume of 20 µL. The cycling conditions were as follows: one cycle at 95°C for 2 min, followed by 30 cycles of 95°C for 20 sec, 58°C for 30 sec, and 72°C for 30 sec. The resulting product was precipitated with ethanol for buffer exchange and was digested at 37°C for overnight in 10 µL of the solution containing 1× L buffer and 2 units of the AluI restriction enzyme. After inactivation at 80°C for 10 min, the digested fragments were analyzed using a microchip electrophoresis system (MCE-202 MultiNA; Shimazu) with the DNA-500 reagent kit. The molar concentrations of both digested and undigested fragments were quantified using the MultiNA Viewer software. The mutation rate was calculated as the ratio of the undigested fragment to the sum of the undigested fragment and the larger digested fragment as described previously.

14Potential off-target sites in the medaka genome were searched using a “Pattern Match” tool in New Medaka Map (beta) at the NBRP medaka web site (http://viewer.shigen.info/medakavw/patternmatch) with 2 criteria: criterion (i) perfect matching in the 12-bp sequence at the 3′ end of the 20-bp target sequence and the NGG PAM sequence; criterion (ii) matching of 16 to 18 bp of the 18 bp sequence at the 3′ end of the target followed by either the NGG or the NAG PAM sequence. All identified potential off-target sites were analyzed by HMA using the primers listed in supplementary material Tables S3 and S4, as described above. Subsequently, the genomic region containing altered off-target sites was amplified with TaKaRa Ex Taq (Takara) and subcloned into either the pGEM-T vector (Promega) or the T-Vector pMD20 (Takara). The fragment containing the cloned genomic sequence was amplified from each colony with the M13 forward and reverse primers (supplementary material Table S1), and then each fragment was sequenced using a T7 promoter primer or a SP6 promoter primer (supplementary material Table S1).

15RGEN-injected fish were mated with wild-type fish of the d-rR strain and genomic DNA was extracted from each F1 embryo. Mutation in each embryo was analyzed by HMA using the primers DJ1-FW2 and DJ1-RV2 (supplementary material Table S1). Mutant alleles in each embryo were determined by direct sequencing of the DJ-1 gene region, amplified using the primers DJ1-FW2 and DJ1-RV1 (supplementary material Table S1).

16Mutation rates were analyzed with one-way ANOVAs followed by Tukey's HSD using the R language (http://www.r-project.org).

17To produce customized guide RNAs, we used the pDR274 vector, a T7 polymerase-mediated expression vector for a synthetic sgRNA, that was used in a zebrafish study. First, we designed an sgRNA in the second exon of the DJ-1/park7 gene (Ensembl gene no. ENSORLG00000004285), successfully disrupted by TALENs in our previous study. A sequence of the form 5′-GG-N18-NGG-3′ was selected for the first target (5′-GGCCTCTTCCAAGCTAGTATCGG-3′; site no. 1) according to a previously described design guideline (Fig. 1A,B). To induce efficient expression of Cas9 nuclease, we generated a pCS2+-based Cas9 nuclease expression vector to produce a capped RNA by SP6 RNA polymerase, containing a human codon-optimized S. pyogenes Cas9 nuclease fused to a triple FLAG tag and two nuclear localization signal (NLS) in both N- and C-terminals previously used in cultured human cells.

18To investigate the efficiency of inducing targeted mutations in medaka, the solution containing 25 ng/µL of sgRNA and 100 ng/µL of Cas9 RNA was injected into fertilized eggs of the d-rR medaka strain. Genomic DNA was extracted from each injected embryo at 3 dpf, followed by assessment of the presence of targeted mutations by HMA. Formation of heteroduplexes was observed in all analyzed embryos (Fig. 1C), indicating that the RGEN-mediated indels were induced at the target locus. Subsequently, the DJ-1 gene region containing the target site was PCR-amplified from two representative embryos. The PCR products were subcloned and each clone was sequenced. All the 33 sequenced clones had altered sequences, including 6 types of mutations in embryo #1-1 (17 of 17 sequenced clones; 100%) (Fig. 1D) and 7 types of mutations in embryo #1-2 (16 of 16; 100%) (Fig. 1E). These results indicate that the RGEN introduced DNA double-strand breaks at the target genomic sequence and thereby induced indels via error-prone nonhomologous end joining repair with high efficiency.

19For more flexible targeting by the CRISPR/Cas system, we examined whether GG at the 5′ end of the targeting sequence is required for the sgRNAs transcribed by T7 RNA polymerase. We selected 2 genomic sequences (20 bp) followed by a PAM sequence on the second exon of the DJ-1 gene as additional targets (Fig. 2A). Site no. 2 (5′-CGTCCAGTGCAGCAGAAACGTGG-3′) contains CG at the 5′ end and site no. 3 (5′-CATCTGTCCCGATACTAGCTTGG-3′) contains CA at the 5′ end. To design sgRNAs that target these sequences, we employed 2 strategies as follows: (a) customizing only 18 nts of the sgRNA by replacing mismatches between GG added at the 5′ end and the genomic target sequence; (b) customizing all modifiable 20-nt-long sequences of the sgRNA by ignoring the GG added at the 5′ end. The sgRNA no. 2a or 2b for target site no. 2 (Fig. 2B) and the sgRNA no. 3a or 3b for site no. 3 (Fig. 2C) were designed according to the strategy (a) or (b), respectively. We injected the solution containing 25 ng/µL sgRNA and 100 ng/µL Cas9 RNA. Results of HMA using RGEN-injected embryos showed that all 4 sgRNAs induced mutations at their targeting site (Fig. 2D). Subsequent sequence analysis using 2 representative embryos in each sgRNA not only revealed the introduction of indels, but also exhibited that the efficiency of inducing mutation depended on the design of the sgRNA (Fig. 2E–H). sgRNA no. 2a (Fig. 2E) and 3a (Fig. 2G) induced mutations with high efficiencies (28/28; 100% and 24/24; 100%, respectively), whereas sgRNA no. 2b (Fig. 2F) and 3b (Fig. 2H) induced mutations with relatively lower efficiencies (21/23; 91.3% and 13/29; 44.8%, respectively). These results exhibit that the GG at the 5′ end of the target genomic sequence is not essential for DNA cleavage by the sgRNAs transcribed by the T7 promoter. Additionally, these results indicate that the sgRNAs customized with the 18-nt sequences following the first added GG can induce targeted mutations with higher efficiency.

20To investigate the dose dependence of RGEN-induced mutation, we injected varying amounts of Cas9 RNA and sgRNA no. 3a because its targeting sequence contains an AluI restriction enzyme site that facilitates restriction fragment length polymorphism (RFLP) analysis (Fig. 3A). Most embryos injected with Cas9 RNA and/or the sgRNA developed normally and similar to the untreated embryos (Table 1). Genomic DNA was extracted from each surviving embryo at 3 dpf. Subsequently, the PCR amplicon, which included the targeted genomic sequence, was subjected to AluI digestion and analyzed by the MultiNA system. All samples that were injected with both Cas9 RNA and sgRNA showed the undigested fragment (a+b, Fig. 3B) while control samples without Cas9 RNA and/or sgRNA showed no undigested fragment but two AluI-digested fragment (a and b, Fig. 3B). Then, we calculated the disrupting activity of the RGEN at each concentration using the quantities of digested and undigested fragments. Serial dilutions of the Cas9 RNA exhibited that 10 ng/µL Cas9 RNA induced mutations with significantly lower efficiency (81.2±2.9%) as compared to 30 and 100 ng/µL (92.4±1.8% and 95.7±2.5%, respectively) (Fig. 3C). Similar results were obtained with sgRNA, as exhibited by a significantly low efficiency of mutation with 1 ng/µL sgRNA (46.4±6.6%) as compared to 10 and 25 ng/µL (96.5±1.3% and 95.7±2.5%, respectively) (Fig. 3D). These results indicate that the efficiency of both Cas9 RNA and sgRNA in inducing mutations is dose dependent.

21To assess the possibility of induction of off-target mutagenesis by RGEN we searched candidate off-target sequences that could potentially be targeted by the 3 sgRNAs (no. 1, 2a, and 3a) for the DJ-1 gene. Previous in vitro studies and in bacteria and human cells have shown that cleavage by Cas9 can be abolished by single mismatches in the “seed” sequence, a 10–12-bp sequence located in the 3′ end of the 20-bp targeting region. We therefore searched the medaka genome for candidate sites that perfectly match the 12-bp sequence at the 3′ ends of the 20-bp targeting sequence and the NGG PAM sequence, referred to as criterion (i), (see “Off-target analysis” in Materials and Methods). We identified 4, 17, and 4 candidate sites for the sgRNA no. 1, 2a, and 3a, respectively (supplementary material Table S3). Using HMA, we detected efficient alterations in the embryos injected with the sgRNA no. 2a at a genomic locus (OT2-I4: 5′-AGTCTAGAGCAGCAGAAACGGGG-3′) harboring 3-bp mismatches (Fig. 4A), while no alteration was detected at other candidate sites (supplementary material Fig. S1).

22Recently, Fu et al. revealed that one or more mismatches located in the 3′ half of the sgRNA targeting region are tolerated, and Hsu et al. also revealed that S. pyogenes Cas9 can cleave targets with a NAG PAM. Therefore, we investigated potential off-target sites identified by another criterion. Based on our data described in the section “The sgRNAs transcribed by T7 polymerase do not necessarily require the target sites starting with GG”, which suggests that the 2-nt sequence at the 5′ end of the sgRNAs is not crucial to targeting sequence recognition of the RGENs, we searched the genome for candidate sites that match with the 18-bp sequence at the 3′ ends of the targeting sequence harboring up to 2-bp mismatches adjacent to either NGG or NAG PAM sequence, referred to as criterion (ii). We identified 4 and 8 additional candidate sites for the target site no. 1 and 2a, respectively (supplementary material Table S4), including the OT2-I4 meeting the first criterion (supplementary material Table S3), and then detected alterations in the embryos injected with the sgRNA no. 2a at a locus (OT2-II4: 5′-AATTCAGTGCAACAGAAACGGGG-3′) (Fig. 4B). DNA sequencing confirmed that mutations were induced with high efficiency at the OT2-I4 (11/11; 100%) (Fig. 4C) and low efficiency at the OT2-II4 (1/10; 10%) (Fig. 4D). These results showed that the RGENs have the potential to induce off-target mutations in vivo.

23Furthermore, we investigated the dose effect of sgRNA on off-target alterations using sgRNA no. 2a. Cas9 RNA (100 ng/µL) was injected with 5 or 10 ng/µL of sgRNA no. 2a and then we analyzed the DJ-1 target locus and two off-target loci (OT2-I4 and OT2-II4), which were mutated in the previous experiment, using genomic DNA mixture from 12 embryos at 3 dpf. At the DJ-1 locus, the lower doses of the sgRNA, 10 ng/µL (12/12; 100%) (Fig. 5B) and 5 ng/µL (8/12; 66.7%) (Fig. 5C), induced mutations as efficiently as 25 ng/µL (28/28; 100%) (Fig. 2E). On the other hand, at the off-target loci OT2-I4 and OT2-II4, the lower doses of the sgRNA, 10 ng/µL (5/11; 45.5% and 0/12; 0%, respectively) (Fig. 5D,F) and 5 ng/µL (1/12; 8.3% and 0/9; 0%, respectively) (Fig. 5E,G), dramatically reduced mutation frequencies compared to 25 ng/µL (11/11; 100% and 1/10; 10%, respectively) (Fig. 4C,D). These results showed that the sgRNA no. 2a induced mutations more efficiently at the on-target locus than the off-target loci. These also suggest that lower dosage of sgRNA is likely to reduce off-target effects by the RGENs.

24To test whether RGENs can induce heritable mutations, we raised the RGEN-injected fish to sexual maturity and analyzed their progeny. Of the 40 eggs injected with 100 ng/µL Cas9 RNA and 25 ng/µL sgRNA no. 1, 32 (80%) hatched normally. Five G0 fish were mated with wild-type fish of the d-rR strain, and their F1 embryos were genotyped by HMA. We identified that all 5 G0 fish transmitted the RGEN-induced mutations to their progeny. The germ line transmission rates of the mutations in each G0 fish ranged from 42.9% (12 of 28; #1–4) to 100% (27 of 27; #1–3, 23 of 23; #1–5) (Fig. 6). The mutation pattern found in the germ cells of each G0 founder varied from 1 (#1–4) to 6 (#1–2). All mutations identified in F1 embryos are shown in Fig. 6. These results indicate that the RGENs induced heritable mutations with high efficiency.

25In this study, we described a simple and efficient approach for targeted mutagenesis in medaka by using the CRISPR/Cas-mediated RGENs. The RGENs can induce targeted genomic cleavage when injected with only capped RNA encoding a Cas9 endonuclease and a sgRNA guiding the nuclease to the complementary genomic sequence. All the designed sgRNAs induced targeted somatic mutations with high efficiency (44.8–100%; average, 86.8%). G0 founders that were injected with the RGENs carried mutations in their germ cells with high efficiency. These efficiencies are as high as those in our mutagenesis study using TALENs in medaka. These results mean that the RGENs function as an efficient engineered nuclease system in medaka.

26The previously designed guidelines for an sgRNA transcribed by T7 RNA polymerase requires any sequence of the form 5′-GG-N18-NGG-3′ that occurs once in every 128 bp of a random DNA sequence. Efficient induction of mutations by the sgRNAs designed for the target site no. 2 and 3 has revealed that mismatches between the 2-nt sequence at the 5′ ends of the sgRNAs and the target genomic sequence are tolerated. It was also reported that double mismatches at the 5′ ends are tolerated in zebrafish, suggesting that the RGEN can target any sequence only adjacent to a NGG PAM sequence that occurs once in every 8 bp. In this study, the sgRNAs starting with the sequence 5′-GG-N18-3′ (no. 2a and 3a) induced mutations more efficiently as compared to the sequence 5′-GG-N20-3′ (no. 2b and 3b). From the results, we propose the guideline for design of sgRNAs that can efficiently disrupt target genomic sequences in medaka (Table 2). On the other hand, one of the 3 examined sgRNA with the sequence 5′-GG-N20-3′ more efficiently induced mutations as compared to the sequence 5′-GG-N18-3′ in the zebrafish study. Since the effects of the 5′ ends of sgRNAs on cleaving activities were investigated in only a few examples, more comprehensive investigations will be required to generalize the effectiveness of the 5′ structures to other targeting sites.

27Previous in vivo works defined a 12-bp sequence adjacent to a PAM as a seed sequence and counted the genomic sequences that perfectly matched the seed sequence as potential off-target sites. However, the study in human cells revealed that one or more mismatches located in the 12-bp sequences are tolerated, suggesting that some in vivo off-target alterations are likely to be missed in these works. In fact, although one of the 2 mutagenized off-target loci (OT2-I4, Fig. 4) meets this criterion (called “criterion (i)” in this study), the other locus (OT2-II4, Fig. 4), harboring a single mismatch in the 12-bp sequence of a seed sequence, does not meet this criterion. Both these mutagenized off-target loci meet the criterion (ii), matching 16 to 18 of 18-bp sequence at the 3′ end of the targeting sequence followed by a NRG (either the NGG or NAG) PAM.

28It was reported that potential off-targets meeting the criterion (ii) were efficiently disrupted in cultured human cells and zebrafish. Altogether, we propose that potential off-target loci identified by the criterion (ii), harboring 2- or fewer bp mismatches in the 18-bp targeting sequence followed by a NRG PAM, should be analyzed for screening unwanted mutagenesis. However, Cho et al. showed that some RGENs can distinguish on-target sites from off-target sites that differ by at least two bases. Both this report and our limited analysis suggest that further investigations (e.g. genome-wide or deep-sequencing analysis) will be required to determine the generalized criteria for searching the off-target sites in medaka and other organisms.

29Significant induction of in vivo off-target alterations indicates that the potentially confounding effects of off-target mutations should be considered in analysis using the RGEN-mediated genome-edited organisms. Also, since our approach for searching of potential off-target sites is based on similarities to the targeting sequences, we may miss some off-target effects in this study. However, in this study, fish injected with the RGENs developed normally (Table 1), suggesting that their off-target effects are not crucial for viability and have little non-specific toxicity unlike found in the fish injected with ZFNs. Additionally, each sgRNA that was designed in this study has unique profile of off-target activities and only the 2 loci for sgRNA no. 2 were mutagenized (Fig. 4; supplementary material Fig. S1). These suggest that validating the phenotypes with at least two independent lines generated by different sgRNAs is important to eliminate the off-target effects, in addition to cleaning the background mutations by repeated outcrossing and rescue experiments. Furthermore, we found that 5 or 10 ng/µL of sgRNA no. 2a induced off-target alterations with significantly lower efficiencies than did 25 ng/µL of the sgRNA (Fig. 5). This indicates that injection with lower dosage of sgRNAs will also become an effective way to reduce the off-target effects.

30At present, ZFNs, TALENs, and CRISPR/Cas-mediated RGENs are 3 successful options for targeted genome editing in medaka. Both the TALEN and the CRISPR/Cas systems allow for the construction of highly efficient nucleases that target desired sequences more easily than the ZFN system. Compared to the TALENs, the CRISPR/Cas system has some advantageous characteristics for efficient genome editing. Firstly, determination of DNA cleavage specificity by guide RNA sequences allows the customizing of the RGENs only by modification of the 5′ end of the sgRNA using annealed oligonucleotides, while the customizing of TALENs requires preparation of multiple vectors (at least 35 vectors, as described earlier) and complex assembly processes. Secondly, the RGENs can efficiently cleave DNA targets containing CpG methylation sites, while TALENs are sensitive to methylation. Finally, Cas9 nickases can be simply engineered by the introduction of a point mutation into Cas9 nuclease (e.g. D10A), which are likely to efficiently induce precise genome engineering via homology-directed repair without the effects of indel mutations induced via non-homologous end joining pathway. These indicate that the CRISPR/Cas-mediated RGENs have the potential to be developed as an alternative technology for targeted genome editing in medaka and other organisms.