For the emerging amphibian genetic model Xenopus tropicalis targeted gene disruption is dependent on zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), which require either complex design and selection or laborious construction. Thus, easy and efficient genome editing tools are still highly desirable for this species. Here, we report that RNA-guided Cas9 nuclease resulted in precise targeted gene disruption in all ten X. tropicalis genes that we analyzed, with efficiencies above 45% and readily up to 100%. Systematic point mutation analyses in two loci revealed that perfect matches between the spacer and the protospacer sequences proximal to the protospacer adjacent motif (PAM) were essential for Cas9 to cleave the target sites in the X. tropicalis genome. Further study showed that the Cas9 system could serve as an efficient tool for multiplexed genome engineering in Xenopus embryos. Analysis of the disruption of two genes, ptf1a/p48 and tyrosinase, indicated that Cas9-mediated gene targeting can facilitate direct phenotypic assessment in X. tropicalis embryos. Finally, five founder frogs from targeting of either elastase-T1, elastase-T2 or tyrosinase showed highly efficient transmission of targeted mutations into F1 embryos. Together, our data demonstrate that the Cas9 system is an easy, efficient and reliable tool for multiplex genome editing in X. tropicalis.

INTRODUCTION

Bacterial and archaeal clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) adaptive immune systems rely on small RNAs in complex with Cas proteins to silence foreign nucleic acids, including viruses and plasmids. There are three major types of CRISPR/Cas systems (Makarova et al., 2011; Wiedenheft et al., 2012). In the type II CRISPR system, the Cas9 protein forms a complex with two short non-coding RNAs, namely the spacer-containing RNA (crRNA) and the trans-activating CRISPR RNA (tracrRNA), to selectively cleave the invading DNA. With the recapitulation of this DNA cleavage activity in vitro with purified Cas9 and an engineered single guide RNA (gRNA) molecule containing the minimal features of both spacer and tracr RNAs (Jinek et al., 2012), it was anticipated that the system could potentially be used in place of zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) for targeted genomic cleavage in higher organisms (Carroll, 2012; Jinek et al., 2012). Indeed, it has been shown that, via generation of site-specific DNA double-strand breaks in the target loci, RNA-guided Cas9 nuclease facilitates genome editing in yeast, nematode, fly, zebrafish and mice, in mouse and human cell lines, as well as in plants (Bassett et al., 2013; Chang et al., 2013; Cho et al., 2013; Cong et al., 2013; DiCarlo et al., 2013; Dickinson et al., 2013; Ding et al., 2013; Fujii et al., 2013; Gratz et al., 2013; Hwang et al., 2013; Jiang et al., 2013; Mali et al., 2013; Shen et al., 2013; Wang et al., 2013; Yang et al., 2013).

In the past decade, the diploid frog Xenopus tropicalis has emerged as an excellent amphibian genetic model (Harland and Grainger, 2011). We and others have established ZFN- or TALEN-mediated gene targeting protocols in this species (Ishibashi et al., 2012; Lei et al., 2012; Young et al., 2011). In comparison to ZFNs, TALENs are more effective in frogs. Despite the ease of designing TALE modules, it is by no means trivial to generate TALENs in the laboratory for use in large-scale reverse genetics. Thus, efficient genome engineering tools that can be easily and cost-effectively generated are still highly desirable. Here, we report that gRNA/Cas9 can serve as an easy, economic, efficient and reliable tool for targeted gene disruption in X. tropicalis.

RESULTS

Optimization of gRNA and Cas9 doses in X. tropicalis embryos

First, we injected Cas9 mRNA at a dose of 500 pg per embryo together with gRNAs (50 pg/embryo) targeting ptf1a/p48, hhex or pat into one-cell stage X. tropicalis embryos. All three injection groups showed high levels of dead and deformed embryos (Fig. 1A,B), indicating non-specific toxicity. We then chose hhex and pat gRNAs to optimize the doses of Cas9 mRNA and gRNA for X. tropicalis embryos based purely on the morphological phenotype. The data obtained indicate that the optimal Cas9 mRNA dose is 300 pg/embryo (Fig. 1C) and the quantity of gRNA should not exceed 500 pg/embryo (Fig. 1D,E). In all subsequent experiments, we set the Cas9 mRNA dose at 300 pg/embryo; for a given locus, the gRNA dose was further optimized in the range 1-500 pg per embryo.

Fig. 1.

The optimal dose of Cas9 mRNA for X. tropicalis embryos is 300 pg/embryo and the quantity of gRNA should not exceed 500 pg/embryo. (A) Representative morphology of dead and abnormal embryos evaluated when control siblings reached stage 30. The deformation was mainly caused by gastrulation defects. (B) Cas9 mRNA at 500 pg/embryo appeared toxic to X. tropicalis embryos. Dead and abnormal embryos were scored upon injection of Cas9 mRNA (500 pg/embryo) and gRNA (50 pg/embryo) targeting the indicated genes. (C) Constant amount (50 pg/embryo) of gRNA targeting hhex and graded doses of Cas9 mRNA (in pg/embryo) were injected into one-cell stage X. tropicalis embryos and the resulting dead and abnormal embryos were scored. (D,E) Constant amount of Cas9 mRNA (300 pg/embryo) and various doses (in pg/embryo) of gRNAs targeting either hhex (D) or pat (E) were injected into one-cell stage X. tropicalis embryos and the resulting dead and abnormal embryos were scored. (B-E) The total embryos for each injection (n) is given under each column.

Fig. 1.

The optimal dose of Cas9 mRNA for X. tropicalis embryos is 300 pg/embryo and the quantity of gRNA should not exceed 500 pg/embryo. (A) Representative morphology of dead and abnormal embryos evaluated when control siblings reached stage 30. The deformation was mainly caused by gastrulation defects. (B) Cas9 mRNA at 500 pg/embryo appeared toxic to X. tropicalis embryos. Dead and abnormal embryos were scored upon injection of Cas9 mRNA (500 pg/embryo) and gRNA (50 pg/embryo) targeting the indicated genes. (C) Constant amount (50 pg/embryo) of gRNA targeting hhex and graded doses of Cas9 mRNA (in pg/embryo) were injected into one-cell stage X. tropicalis embryos and the resulting dead and abnormal embryos were scored. (D,E) Constant amount of Cas9 mRNA (300 pg/embryo) and various doses (in pg/embryo) of gRNAs targeting either hhex (D) or pat (E) were injected into one-cell stage X. tropicalis embryos and the resulting dead and abnormal embryos were scored. (B-E) The total embryos for each injection (n) is given under each column.

gRNA/Cas9 is an efficient and reliable tool for genome editing in X. tropicalis

We initially designed gRNAs targeting 12 loci in ten different genes (Table 1; supplementary material Table S1). Those targeting elastase-T1, ets2, tm4sf4-T2, grp78, elastase-T2 and ptf1a/p48 readily exhibited targeting efficiencies above 72% at a dose of 50 pg/embryo and the first three even achieved 100% efficiency (Fig. 2A; supplementary material Fig. S1). The mutagenesis rates induced by gRNAs targeting hhex, tm4sf4-T1 and tyrosinase were raised from 31.3%, 60% and 60% to 100%, 86.7% and 82.4% when the gRNA doses were increased from an initial 50 pg/embryo to 500, 200 and 400 pg/embryo, respectively (Fig. 2A-D; supplementary material Fig. S1). The highest efficiency obtained for ets1 gRNA was 33.3%, and gRNAs targeting pat and pdx1 showed either very low efficiency or no effect at the various gRNA doses tested (Fig. 2A,E-G; supplementary material Fig. S1). We then designed two additional gRNAs for each of ets1, pat and pdx1. The data obtained indicate that all caused mutations with high efficiencies (45-79%) at 50 pg/embryo (Fig. 2H; supplementary material Fig. S2). Thus, all of the genes tested were readily targeted by gRNA/Cas9 with efficiencies above 45%. Finally, we chose one of the most effective gRNAs to scale down the gRNA dose, and found that 15 pg/embryo of elastase-T1 gRNA still exhibited 83.3% efficiency (Fig. 2I). Together, our results strongly suggest that gRNA/Cas9 can efficiently target most loci in the X. tropicalis genome.

Fig. 2.

gRNA/Cas9 is an efficient and robust tool for gene targeting in X. tropicalis. (A) gRNA/Cas9 induced efficient targeted gene disruption in X. tropicalis embryos. The genes targeted and the doses of gRNAs (in pg/embryo) used are indicated. The dose of Cas9 mRNA was set at 300 pg/embryo for all injections in this figure. (B-G) Targeting efficiencies can be improved by increasing the amount of gRNA (pg/embryo shown), as evaluated with a constant amount of Cas9 mRNA (300 pg/embryo). (H) The targeting efficiencies of further gRNAs (50 pg/embryo). (I) At 15 pg/embryo, elastase-T1 gRNA was still 83.3% efficient. The numbers above each bar indicate mutations detected among total samples sequenced.

Fig. 2.

gRNA/Cas9 is an efficient and robust tool for gene targeting in X. tropicalis. (A) gRNA/Cas9 induced efficient targeted gene disruption in X. tropicalis embryos. The genes targeted and the doses of gRNAs (in pg/embryo) used are indicated. The dose of Cas9 mRNA was set at 300 pg/embryo for all injections in this figure. (B-G) Targeting efficiencies can be improved by increasing the amount of gRNA (pg/embryo shown), as evaluated with a constant amount of Cas9 mRNA (300 pg/embryo). (H) The targeting efficiencies of further gRNAs (50 pg/embryo). (I) At 15 pg/embryo, elastase-T1 gRNA was still 83.3% efficient. The numbers above each bar indicate mutations detected among total samples sequenced.

Table 1.

The 18 targeting loci in ten X. tropicalis genes and the oligonucleotides used to construct the corresponding gRNA constructs

The 18 targeting loci in ten X. tropicalis genes and the oligonucleotides used to construct the corresponding gRNA constructs
The 18 targeting loci in ten X. tropicalis genes and the oligonucleotides used to construct the corresponding gRNA constructs

A perfect match between the spacer and protospacer sequences proximal to the PAM is essential for Cas9 to cleave target DNA in the X. tropicalis genome

To investigate the specificity of the gRNA/Cas9 system for genome editing in whole organisms, we chose two loci (ets2 and tm4sf4-T2) that displayed 100% targeting efficiency and systematically analyzed the consequence of single-nucleotide mismatches between the spacer and the protospacer sequences for targeting efficiency in X. tropicalis embryos. For both genes, point mutations up to the eleventh base pair upstream of the protospacer adjacent motif (PAM) completely abolished the targeting activity of gRNA/Cas9. By contrast, gRNA/Cas9-mediated target cleavage is partially tolerant to point mutations 12, 13, 15, 17, 18, 19 and 20 bp 5′ of the PAM (Fig. 3; supplementary material Figs S3 and S4).

Fig. 3.

A perfect match between the spacer and the protospacer sequences proximal to the PAM is essential for Cas9 to cleave target sites in the X. tropicalis genome. (A,B) ets2- or tm4sf4-T2-targeting crRNAs containing single-point mutations (red) were generated to investigate the consequences of single-nucleotide mismatches between the spacer and the protospacer sequences for Cas9-mediated gene targeting efficiency in X. tropicalis embryos. The targeting efficiency is indicated on the right of each mutant. The PAM sequence is indicated (blue). wt, wild type.

Fig. 3.

A perfect match between the spacer and the protospacer sequences proximal to the PAM is essential for Cas9 to cleave target sites in the X. tropicalis genome. (A,B) ets2- or tm4sf4-T2-targeting crRNAs containing single-point mutations (red) were generated to investigate the consequences of single-nucleotide mismatches between the spacer and the protospacer sequences for Cas9-mediated gene targeting efficiency in X. tropicalis embryos. The targeting efficiency is indicated on the right of each mutant. The PAM sequence is indicated (blue). wt, wild type.

To further assess whether gRNA/Cas9 creates any off-target mutations in frog embryos, we first computationally identified all the potential off-target sites with up to five mismatches to all the loci targeted in this study (supplementary material Table S2). Since no sites with one mismatch were identified, we selected 119 sites in total, including all four sites with two mismatches, seven sites with three mismatches distal to the PAM sequence, and all sites with up to four mismatches for the ets2, ptf1a/p48 and tyrosinase target loci, and performed a T7EI assay to identify any off-target disruptions (supplementary material Table S3). In contrast to the on-target sites, no potential off-target sites analyzed showed reliable gRNA/Cas9-dependent T7EI assay positive signals (data not shown). Our study suggests that the frequency of cleavage within potential off-target sites with two to four mismatches is too low to be detected by our T7EI assay.

Multiplexed gene targeting in X. tropicalis

To test whether this approach is suitable for multiplexed editing of genomic loci in Xenopus embryos, we co-injected Cas9 mRNA together with two gRNAs targeting grp78 and elastase-T1. The data indicate that the targeting efficiencies for each gene from the co-injection are almost identical to those obtained from the individual injections (Fig. 4A; supplementary material Fig. S5). Single-cell analysis for stage 9 embryos (blastulae) indicates that both alleles of the two loci targeted were mutated in the same cell (Fig. 4B,C). Given the high targeting efficiency in the founder embryos and high germ line transmission rates observed in this study with other genes, these data suggest that double or triple knockout lines of genes of interest in X. tropicalis could be established from a single injection of Cas9/gRNAs, which also appears to be achievable in mice (Wang et al., 2013).

Fig. 4.

gRNA/Cas9 is suitable for multiplexed genome editing in X. tropicalis. (A) Co-injection of Cas9 mRNA (300 pg/embryo) together with two gRNAs targeting grp78 (grp) and elastase-T1 (ela) did not affect the targeting efficiencies obtained from individual injections. The gRNA dose for each gene was set at 50 pg/embryo. (B,C) DNA sequencing data obtained from the progenies of two different blastomeres (shown separately in B and C) of a stage 9 embryo demonstrate that both loci were disrupted in the same cell. The wild-type sequence is shown at the top with the target site highlighted in yellow and the PAM sequence in blue. Red dashes indicate deletions and lowercase letters in red indicate insertions. The number of deleted (Δ) or inserted (+) base pairs is indicated in parentheses; numbers in square brackets show the frequencies of the mutation among the sequenced samples. The data indicate that both alleles of both loci were mutated in progenies of each blastomere analyzed.

Fig. 4.

gRNA/Cas9 is suitable for multiplexed genome editing in X. tropicalis. (A) Co-injection of Cas9 mRNA (300 pg/embryo) together with two gRNAs targeting grp78 (grp) and elastase-T1 (ela) did not affect the targeting efficiencies obtained from individual injections. The gRNA dose for each gene was set at 50 pg/embryo. (B,C) DNA sequencing data obtained from the progenies of two different blastomeres (shown separately in B and C) of a stage 9 embryo demonstrate that both loci were disrupted in the same cell. The wild-type sequence is shown at the top with the target site highlighted in yellow and the PAM sequence in blue. Red dashes indicate deletions and lowercase letters in red indicate insertions. The number of deleted (Δ) or inserted (+) base pairs is indicated in parentheses; numbers in square brackets show the frequencies of the mutation among the sequenced samples. The data indicate that both alleles of both loci were mutated in progenies of each blastomere analyzed.

Phenotyping of gRNA/Cas9-targeted G0 embryos, froglets and frogs

In principle, the high efficiency of gene disruption induced by Cas9 nuclease could allow for direct phenotype assessment in gRNA/Cas9-injected Xenopus embryos. Our data indicated that the expression of the pancreas-specific marker gene pdip is indeed completely inhibited in a portion of ptf1a/p48 gRNA-injected embryos (Fig. 5A,E). The rest of the targeted embryos showed severe inhibition of pdip expression (Fig. 5B,F), with hardly any showing the strong signals seen in wild-type or elastase-targeted embryos (Fig. 5I-L). Co-injection of a dexamethasone-inducible variant of Ptf1a/p48 (Ptf1a/p48GR), which was activated after gastrulation, resulted in 100% rescue of the ptf1a/p48 gRNA-induced phenotype (Fig. 5D,H). Together, these findings are reminiscent of those obtained upon application of ptf1a/p48 morpholinos to Xenopus laevis embryos (Afelik et al., 2006; Jarikji et al., 2007). We also dissected six ptf1a/p48-targeted froglets that all showed severe pancreatic hypoplasia (Fig. 5N), consistent with our previous findings with ptf1a/p48 TALENs (Lei et al., 2012).

Fig. 5.

gRNA/Cas9-mediated gene targeting is suitable for G0 phenotyping. (A-L) Whole-mount in situ hybridization analysis of expression of the pancreas-specific marker pdip in X. tropicalis normal control tadpoles (stage 40), tadpoles injected with Cas9 mRNA (300 pg/embryo) and gRNAs (50 pg/embryo), and tadpoles injected with either ptf1a/p48GR mRNA (20 pg/embryo) alone or in combination with Cas9 and ptf1a/p48 gRNA. Dexamethasone (working concentration of 10 μM) was added at stage 14 to activate Ptf1a/p48. (A,E) Complete inhibition of pdip expression upon targeting ptf1a/p48. (B,F) Partial inhibition of pdip expression upon targeting ptf1a/p48. (C,G) Overexpression of ptf1a/p48 expands pdip expression in the territory of the stomach and duodenum. (D,H) The inhibition of pdip expression upon gRNA/Cas9-mediated targeting of ptf1a/p48 was completely rescued by co-injection of ptf1a/p48GR mRNA. (I,K) Uninjected control embryos. (J,L) As a negative control, pdip expression was unaffected upon targeting elastase. All the images are lateral views with the head to the left. E-H,K,L show further examples of the types represented in A-D,I,J, respectively. The number of embryos showing the illustrated phenotype is given in the representative image. (M,N) Dissection of 1-week-old froglets revealed severe pancreatic hypoplasia in ptf1a/p48 gRNA/Cas9-injected G0 froglets, with stomach and duodenum unaffected. The pancreas is outlined (dashed line). du, duodenum; st, stomach. (O-V) Albinism phenotype caused by tyrosinase gRNA/Cas9. (O,S) Uninjected control tadpoles. (P,T) Almost full albinism. (Q,U) Tadpoles showing severe perturbation of pigmentation. (R,V) Partial albinism. S-V show further examples of the types represented in O-R, respectively. The number of embryos showing the illustrated phenotype is given in the representative image. (W-Z) Dorsal view of adult frogs. (W) Wild type. (X) Almost full albinism caused by tyrosinase gRNA/Cas9. (Y,Z) Partial albinism. The numbers of frogs showing the illustrated phenotypes are listed. Scale bars: 400 μm in A-L; 2 mm in M,N; 1 mm on O-V; 1 cm in W-Z.

Fig. 5.

gRNA/Cas9-mediated gene targeting is suitable for G0 phenotyping. (A-L) Whole-mount in situ hybridization analysis of expression of the pancreas-specific marker pdip in X. tropicalis normal control tadpoles (stage 40), tadpoles injected with Cas9 mRNA (300 pg/embryo) and gRNAs (50 pg/embryo), and tadpoles injected with either ptf1a/p48GR mRNA (20 pg/embryo) alone or in combination with Cas9 and ptf1a/p48 gRNA. Dexamethasone (working concentration of 10 μM) was added at stage 14 to activate Ptf1a/p48. (A,E) Complete inhibition of pdip expression upon targeting ptf1a/p48. (B,F) Partial inhibition of pdip expression upon targeting ptf1a/p48. (C,G) Overexpression of ptf1a/p48 expands pdip expression in the territory of the stomach and duodenum. (D,H) The inhibition of pdip expression upon gRNA/Cas9-mediated targeting of ptf1a/p48 was completely rescued by co-injection of ptf1a/p48GR mRNA. (I,K) Uninjected control embryos. (J,L) As a negative control, pdip expression was unaffected upon targeting elastase. All the images are lateral views with the head to the left. E-H,K,L show further examples of the types represented in A-D,I,J, respectively. The number of embryos showing the illustrated phenotype is given in the representative image. (M,N) Dissection of 1-week-old froglets revealed severe pancreatic hypoplasia in ptf1a/p48 gRNA/Cas9-injected G0 froglets, with stomach and duodenum unaffected. The pancreas is outlined (dashed line). du, duodenum; st, stomach. (O-V) Albinism phenotype caused by tyrosinase gRNA/Cas9. (O,S) Uninjected control tadpoles. (P,T) Almost full albinism. (Q,U) Tadpoles showing severe perturbation of pigmentation. (R,V) Partial albinism. S-V show further examples of the types represented in O-R, respectively. The number of embryos showing the illustrated phenotype is given in the representative image. (W-Z) Dorsal view of adult frogs. (W) Wild type. (X) Almost full albinism caused by tyrosinase gRNA/Cas9. (Y,Z) Partial albinism. The numbers of frogs showing the illustrated phenotypes are listed. Scale bars: 400 μm in A-L; 2 mm in M,N; 1 mm on O-V; 1 cm in W-Z.

As a second example, we chose to phenotype the disruption of tyrosinase, which causes the ablation of pigmentation (Beermann et al., 2004; Damé et al., 2012; Ishibashi et al., 2012; Koga et al., 1995; Oetting et al., 2003). Upon tyrosinase gRNA/Cas9 injection, the majority of tadpoles (100/165, ∼61%) showed severe perturbation of pigmentation, with two showing almost full albinism, whereas the remainder displayed partial albinism with none showing the pigmentation pattern seen in wild-type siblings (Fig. 5O-V). The various levels of albinism were maintained to adulthood (Fig. 5X-Z).

gRNA/Cas9-injected founder frogs show high germ line transmission rates

The high efficiency of somatic targeting in gRNA/Cas9-injected embryos would suggest a similarly high targeting efficiency in germ cells of G0 frogs. Just as expected, all five founder male frogs from the targeting of either elastase-T1, elastase-T2 or tyrosinase transmitted their targeted mutations through the germ line with high efficiencies ranging from 40-100% (Fig. 6). The data indicate that gRNA/Cas9-induced mutagenesis in X. tropicalis is highly heritable.

Fig. 6.

gRNA/Cas9-induced targeted mutations are highly heritable. (A-C) DNA sequencing data showing the genotypes of each F1 embryo obtained from founder frogs treated as indicated. The wild-type sequence is shown at the top with the target site highlighted in yellow and the PAM sequence in blue. Red dashes indicate deletions and lowercase letters in red indicate insertions. The number of deleted (Δ) or inserted (+) base pairs is indicated in parentheses; numbers in square brackets show the frequencies of the genotype among the ten sequenced samples.

Fig. 6.

gRNA/Cas9-induced targeted mutations are highly heritable. (A-C) DNA sequencing data showing the genotypes of each F1 embryo obtained from founder frogs treated as indicated. The wild-type sequence is shown at the top with the target site highlighted in yellow and the PAM sequence in blue. Red dashes indicate deletions and lowercase letters in red indicate insertions. The number of deleted (Δ) or inserted (+) base pairs is indicated in parentheses; numbers in square brackets show the frequencies of the genotype among the ten sequenced samples.

DISCUSSION

We have shown that gRNA/Cas9 is an efficient, simple and robust tool for X. tropicalis genome editing with high precision and specificity. Specificity in genome editing is crucial to both basic research and therapeutic application. Our data from the systematic analysis of the effects of single-nucleotide mismatches between the spacer and the protospacer sequences on Cas9-mediated gene targeting efficiencies in X. tropicalis embryos are consistent with findings obtained in vitro and in bacteria and mammalian cell lines (Cong et al., 2013; Fu et al., 2013; Hsu et al., 2013; Jinek et al., 2012; Sapranauskas et al., 2011), further highlighting the importance of the 3′ protospacer sequence close to the PAM in designing gRNAs to eliminate off-target effects. In contrast to the high level of off-target cleavage reported in human cell lines using the CRISPR/Cas system (Cradick et al., 2013; Fu et al., 2013; Hsu et al., 2013), our data suggest that the gRNA/Cas9-induced off-target mutation rate is very low in X. tropicalis embryos, consistent with data obtained with mouse embryos (Yang et al., 2013). Future studies using whole-genome sequencing would generate more comprehensive information. Meanwhile, the use of paired gRNA/Cas9 nickases significantly improves the specificity (Ran et al., 2013).

Sequencing data indicate that both Cas9 and TALEN induce diverse indels (supplementary material Fig. S1) (Lei et al., 2012), suggesting that different embryonic cells are likely to harbor different genotypes of the targeted gene. If some of the mutations do not result in a loss of function, the injected embryos would display a mosaic phenotype. It is also possible that a few cells carry a disruption in only one allele, or even in neither allele. Thus, the mosaicism and monoallelic mutagenesis presumably explain why the sequence disruption efficiency of ptf1a/p48 and tyrosinase was high (72% and 82%, respectively), but the complete inhibition of pdip expression or ablation of pigmentation was low (14% and 1%, respectively); indeed, the rest of the targeted embryos showed intermediate mosaic phenotypes. tyrosinase TALENs also cause mosaic phenotypes in X. tropicalis embryos (Ishibashi et al., 2012). Obviously, the higher the targeting efficiency of the gRNA/Cas9 (especially for those with 100% gene targeting efficiency), the better the opportunity for phenotype assessment in G0 embryos.

Among the 12 gRNAs initially designed, two (∼17%) showed almost no activity. Inactive gRNAs have also been reported for zebrafish embryos (Hwang et al., 2013). We found that when faced with these rare inactive gRNAs, one could easily find active substitutes in the neighboring loci.

In conclusion, our results demonstrate that gRNA/Cas9 is a superb tool for multiplex genome editing in X. tropicalis. Given the simplicity and low cost of gRNA construction, the high targeting efficiency of the gRNA/Cas9 system, the high efficiency of germ line transmission, the relatively short generation time of frogs (4-6 months), and the availability of the frog genome sequence (Hellsten et al., 2010), there is no doubt that the diploid frog X. tropicalis is jumping into the future of developmental genetics.

MATERIALS AND METHODS

Production of Cas9 mRNA, gRNAs and ptf1a/p48 mRNA

The recently reported (Cong et al., 2013) codon-optimized Streptococcus pyogenes Cas9 cDNA together with the two attached nuclear localization signals (3xFLAG-NLS-SpCas9-NLS) was synthesized by GenScript and cloned into the pCS2+ vector (Rupp et al., 1994) (supplementary material Fig. S6). The construct was linearized with NotI and transcribed with the mMessage mMachine SP6 Kit (Ambion) to produce capped Cas9 RNA, which was purified with the RNeasy Mini Kit (Qiagen) according to the RNA clean protocol.

To create a gRNA expression vector, we placed a T7 promoter followed by two BbsI sites upstream of the recently described gRNA scaffold (Mali et al., 2013), which was synthesized by GenScript and cloned into the pUC57-Simple vector (GenScript) (supplementary material Fig. S6). The gRNAs were designed to target protospacer sequences in genes of interest with the form 5′-GG-(N)18-NGG-3′ (Table 1). NGG is the PAM. The locus-specific 20 bp protospacer containing the cloning cohesive sites was obtained by annealing two synthesized partially complementary oligonucleotides (Table 1), and was then cloned into BbsI-digested gRNA expression vector. The resulting construct was digested with DraI and transcribed using the MAXIscript T7 Kit (Ambion). The gRNA was purified by miRNeasy Mini Kit (Qiagen).

Capped ptf1a/p48GR mRNA was generated as described (Afelik et al., 2006).

Manipulation of X. tropicalis embryos and evaluation of gRNA/Cas9-associated toxicity

X. tropicalis frogs were purchased from Nasco. Ovulation and in vitro fertilization were carried out according to the protocol described previously (Khokha et al., 2002; Young et al., 2011). The desired amount of Cas9 mRNA and gRNA in 2 nl was co-injected into one-cell stage embryos. During subsequent development, dead and abnormal embryos (mainly due to gastrulation defects) were sorted out and counted for the purposes of morphological phenotyping.

Evaluation of gene targeting efficiency in gRNA/Cas9-injected embryos

Forty-eight hours after microinjection (about stage 40), we randomly pooled five healthy embryos from each injection, extracted genomic DNA, amplified the targeted region by PCR (for primers see supplementary material Table S4), and then cloned the purified PCR products into the pMD18-T vector (Takara) by TA cloning. Twenty single colonies were randomly picked for DNA sequencing analysis to detect any insertion or deletion (indel) mutations resulting from error-prone non-homologous end joining (NHEJ)-based repair of Cas9-created double-strand breaks. The targeting efficiency was determined by the ratio of mutant to total colonies.

For single-cell analysis, stage 9 embryos co-injected with Cas9 mRNA together with two gRNAs targeting grp78 and elastase-T1 were freed from the vitelline membrane and dissociated in calcium- and magnesium-free medium (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 7.5 mM Tris pH 7.6). Single blastomeres from the dissociated embryos were separately cultured with 1× MBS solution (88 mM NaCl, 2.4 mM NaHCO3, 1 mM KCl, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 10 mM HEPES pH 7.4) in 24-well plates lined with 0.8% agar overnight and then subjected to proteinase K digestion, PCR amplification and TA cloning. Ten single colonies from each blastomere progeny were sequenced to determine the genotype of individual blastomeres.

Measurement of germ line transmission

gRNA/Cas9-injected X. tropicalis embryos were raised to sexual maturity. Male founder frogs were crossed with wild-type females and individual F1 embryos were collected 48 hours postfertilization for genomic DNA extraction. Evaluation of mutations was carried out by PCR amplification, TA cloning and DNA sequencing of single colonies. Ten embryos from each founder frog were analyzed, and for each F1 embryo ten colonies were sequenced.

Identification of potential off-target sites in the X. tropicalis genome

All genomic loci containing up to five mismatches compared with the coding sequence for a given 20 nt gRNA followed by the NGG PAM sequence were identified by mapping the targeted site to X. tropicalis genome V4.1 using a PERL script developed according to the SeqMap method (Jiang and Wong, 2008).

T7 endonuclease I (T7EI) assay for detecting off-target mutagenesis

The T7EI assay was performed essentially as described (Guschin et al., 2010). For each injection, gRNA/Cas9-injected embryos or uninjected control embryos at stage 40 were pooled in groups of five for genomic DNA extraction. The regions of interest containing the off-target sites were amplified by PCR with gene-specific primers (supplementary material Table S3). PCR products were denatured and annealed under the following conditions: 95°C for 5 minutes, 95-85°C at -2°C/s, 85-25°C at -0.1°C/s, hold at 4°C. The annealed samples were digested with T7EI (NEB M0302L), separated and measured on an ethidium bromide-stained 10% polyacrylamide TBE gel and quantified using ImageJ software (NIH).

Whole-mount in situ hybridization

The digoxigenin-labeled antisense X. tropicalis pdip probe was transcribed with T7 RNA polymerase using an RT-PCR-amplified template containing the T7 promoter (forward, 5′-GAGGAGGAGACATCAGACGA-3′; reverse, 5′-CAGTAATACGACTCACTATAGGGAATACTCAAGGACCGAAGAAA-3′). Whole-mount in situ hybridization was performed as described (Harland, 1991).

Acknowledgements

We thank Yinying Long for technical assistance and frog husbandry.

Author contributions

X.G., T.Z., Z.H., H.Z. and Y. Chen designed the work and analyzed experiments. X.G., T.Z., Z.H., Y.Z., Z.S., Y. Cui, and F.W. carried out the experiments. Q.W. designed the PERL script. Y. Chen, T.Z. and X.G. wrote the manuscript.

Funding

This work was supported by the National Basic Research Program of China [2009CB941202 to Y. Chen]; the National Natural Science Foundation of China [31271554 to Y. Chen, 31301192 to X.G.]; and the Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences.

References

Afelik
S.
,
Chen
Y.
,
Pieler
T.
(
2006
).
Combined ectopic expression of Pdx1 and Ptf1a/p48 results in the stable conversion of posterior endoderm into endocrine and exocrine pancreatic tissue.
Genes Dev.
20
,
1441
1446
.
Bassett
A. R.
,
Tibbit
C.
,
Ponting
C. P.
,
Liu
J. L.
(
2013
).
Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system.
Cell Rep.
4
,
220
228
.
Beermann
F.
,
Orlow
S. J.
,
Lamoreux
M. L.
(
2004
).
The Tyr (albino) locus of the laboratory mouse.
Mamm. Genome
15
,
749
758
.
Carroll
D.
(
2012
).
A CRISPR approach to gene targeting.
Mol. Ther.
20
,
1658
1660
.
Chang
N.
,
Sun
C.
,
Gao
L.
,
Zhu
D.
,
Xu
X.
,
Zhu
X.
,
Xiong
J. W.
,
Xi
J. J.
(
2013
).
Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos.
Cell Res.
23
,
465
472
.
Cho
S. W.
,
Kim
S.
,
Kim
J. M.
,
Kim
J. S.
(
2013
).
Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.
Nat. Biotechnol.
31
,
230
232
.
Cong
L.
,
Ran
F. A.
,
Cox
D.
,
Lin
S.
,
Barretto
R.
,
Habib
N.
,
Hsu
P. D.
,
Wu
X.
,
Jiang
W.
,
Marraffini
L. A.
, et al. 
. (
2013
).
Multiplex genome engineering using CRISPR/Cas systems.
Science
339
,
819
823
.
Cradick
T. J.
,
Fine
E. J.
,
Antico
C. J.
,
Bao
G.
(
2013
).
CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity.
Nucleic Acids Res.
41
,
9584
9592
.
Damé
M. C.
,
Xavier
G. M.
,
Oliveira-Filho
J. P.
,
Borges
A. S.
,
Oliveira
H. N.
,
Riet-Correa
F.
,
Schild
A. L.
(
2012
).
A nonsense mutation in the tyrosinase gene causes albinism in water buffalo.
BMC Genet.
13
,
62
.
DiCarlo
J. E.
,
Norville
J. E.
,
Mali
P.
,
Rios
X.
,
Aach
J.
,
Church
G. M.
(
2013
).
Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.
Nucleic Acids Res.
41
,
4336
4343
.
Dickinson
D. J.
,
Ward
J. D.
,
Reiner
D. J.
,
Goldstein
B.
(
2013
).
Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination.
Nat. Methods
10
,
1028
1034
.
Ding
Q.
,
Regan
S. N.
,
Xia
Y.
,
Oostrom
L. A.
,
Cowan
C. A.
,
Musunuru
K.
(
2013
).
Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs.
Cell Stem Cell
12
,
393
394
.
Fu
Y.
,
Foden
J. A.
,
Khayter
C.
,
Maeder
M. L.
,
Reyon
D.
,
Joung
J. K.
,
Sander
J. D.
(
2013
).
High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.
Nat. Biotechnol.
31
,
822
826
.
Fujii
W.
,
Kawasaki
K.
,
Sugiura
K.
,
Naito
K.
(
2013
).
Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease.
Nucleic Acids Res.
41
,
e187
.
Gratz
S. J.
,
Cummings
A. M.
,
Nguyen
J. N.
,
Hamm
D. C.
,
Donohue
L. K.
,
Harrison
M. M.
,
Wildonger
J.
,
O’Connor-Giles
K. M.
(
2013
).
Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease.
Genetics
194
,
1029
1035
.
Guschin
D. Y.
,
Waite
A. J.
,
Katibah
G. E.
,
Miller
J. C.
,
Holmes
M. C.
,
Rebar
E. J.
(
2010
).
A rapid and general assay for monitoring endogenous gene modification.
Methods Mol. Biol.
649
,
247
256
.
Harland
R. M.
(
1991
).
In situ hybridization: an improved whole-mount method for Xenopus embryos.
Methods Cell Biol.
36
,
685
695
.
Harland
R. M.
,
Grainger
R. M.
(
2011
).
Xenopus research: metamorphosed by genetics and genomics.
Trends Genet.
27
,
507
515
.
Hellsten
U.
,
Harland
R. M.
,
Gilchrist
M. J.
,
Hendrix
D.
,
Jurka
J.
,
Kapitonov
V.
,
Ovcharenko
I.
,
Putnam
N. H.
,
Shu
S.
,
Taher
L.
, et al. 
. (
2010
).
The genome of the Western clawed frog Xenopus tropicalis.
Science
328
,
633
636
.
Hsu
P. D.
,
Scott
D. A.
,
Weinstein
J. A.
,
Ran
F. A.
,
Konermann
S.
,
Agarwala
V.
,
Li
Y.
,
Fine
E. J.
,
Wu
X.
,
Shalem
O.
, et al. 
. (
2013
).
DNA targeting specificity of RNA-guided Cas9 nucleases.
Nat. Biotechnol.
31
,
827
832
.
Hwang
W. Y.
,
Fu
Y.
,
Reyon
D.
,
Maeder
M. L.
,
Tsai
S. Q.
,
Sander
J. D.
,
Peterson
R. T.
,
Yeh
J. R.
,
Joung
J. K.
(
2013
).
Efficient genome editing in zebrafish using a CRISPR-Cas system.
Nat. Biotechnol.
31
,
227
229
.
Ishibashi
S.
,
Cliffe
R.
,
Amaya
E.
(
2012
).
Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis.
Biol. Open
1
,
1273
1276
.
Jarikji
Z. H.
,
Vanamala
S.
,
Beck
C. W.
,
Wright
C. V.
,
Leach
S. D.
,
Horb
M. E.
(
2007
).
Differential ability of Ptf1a and Ptf1a-VP16 to convert stomach, duodenum and liver to pancreas.
Dev. Biol.
304
,
786
799
.
Jiang
H.
,
Wong
W. H.
(
2008
).
SeqMap: mapping massive amount of oligonucleotides to the genome.
Bioinformatics
24
,
2395
2396
.
Jiang
W.
,
Zhou
H.
,
Bi
H.
,
Fromm
M.
,
Yang
B.
,
Weeks
D. P.
(
2013
).
Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice.
Nucleic Acids Res.
41
,
e188
.
Jinek
M.
,
Chylinski
K.
,
Fonfara
I.
,
Hauer
M.
,
Doudna
J. A.
,
Charpentier
E.
(
2012
).
A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.
Science
337
,
816
821
.
Khokha
M. K.
,
Chung
C.
,
Bustamante
E. L.
,
Gaw
L. W.
,
Trott
K. A.
,
Yeh
J.
,
Lim
N.
,
Lin
J. C.
,
Taverner
N.
,
Amaya
E.
, et al. 
. (
2002
).
Techniques and probes for the study of Xenopus tropicalis development.
Dev. Dyn.
225
,
499
510
.
Koga
A.
,
Inagaki
H.
,
Bessho
Y.
,
Hori
H.
(
1995
).
Insertion of a novel transposable element in the tyrosinase gene is responsible for an albino mutation in the medaka fish, Oryzias latipes.
Mol. Gen. Genet.
249
,
400
405
.
Lei
Y.
,
Guo
X.
,
Liu
Y.
,
Cao
Y.
,
Deng
Y.
,
Chen
X.
,
Cheng
C. H.
,
Dawid
I. B.
,
Chen
Y.
,
Zhao
H.
(
2012
).
Efficient targeted gene disruption in Xenopus embryos using engineered transcription activator-like effector nucleases (TALENs).
Proc. Natl. Acad. Sci. USA
109
,
17484
17489
.
Makarova
K. S.
,
Haft
D. H.
,
Barrangou
R.
,
Brouns
S. J.
,
Charpentier
E.
,
Horvath
P.
,
Moineau
S.
,
Mojica
F. J.
,
Wolf
Y. I.
,
Yakunin
A. F.
, et al. 
. (
2011
).
Evolution and classification of the CRISPR-Cas systems.
Nat. Rev. Microbiol.
9
,
467
477
.
Mali
P.
,
Yang
L.
,
Esvelt
K. M.
,
Aach
J.
,
Guell
M.
,
DiCarlo
J. E.
,
Norville
J. E.
,
Church
G. M.
(
2013
).
RNA-guided human genome engineering via Cas9.
Science
339
,
823
826
.
Oetting
W. S.
,
Fryer
J. P.
,
Shriram
S.
,
King
R. A.
(
2003
).
Oculocutaneous albinism type 1: the last 100 years.
Pigment Cell Res.
16
,
307
311
.
Ran
F. A.
,
Hsu
P. D.
,
Lin
C. Y.
,
Gootenberg
J. S.
,
Konermann
S.
,
Trevino
A. E.
,
Scott
D. A.
,
Inoue
A.
,
Matoba
S.
,
Zhang
Y.
, et al. 
. (
2013
).
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.
Cell
154
,
1380
1389
.
Rupp
R.
,
Snider
L.
,
Weintraub
H.
(
1994
).
Xenopus embryos regulate the nuclear localization of XMyoD.
Genes Dev.
8
,
1311
1323
.
Sapranauskas
R.
,
Gasiunas
G.
,
Fremaux
C.
,
Barrangou
R.
,
Horvath
P.
,
Siksnys
V.
(
2011
).
The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.
Nucleic Acids Res.
39
,
9275
9282
.
Shen
B.
,
Zhang
J.
,
Wu
H.
,
Wang
J.
,
Ma
K.
,
Li
Z.
,
Zhang
X.
,
Zhang
P.
,
Huang
X.
(
2013
).
Generation of gene-modified mice via Cas9/RNA-mediated gene targeting.
Cell Res.
23
,
720
723
.
Wang
H.
,
Yang
H.
,
Shivalila
C. S.
,
Dawlaty
M. M.
,
Cheng
A. W.
,
Zhang
F.
,
Jaenisch
R.
(
2013
).
One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.
Cell
153
,
910
918
.
Wiedenheft
B.
,
Sternberg
S. H.
,
Doudna
J. A.
(
2012
).
RNA-guided genetic silencing systems in bacteria and archaea.
Nature
482
,
331
338
.
Yang
H.
,
Wang
H.
,
Shivalila
C. S.
,
Cheng
A. W.
,
Shi
L.
,
Jaenisch
R.
(
2013
).
One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.
Cell
154
,
1370
1379
.
Young
J. J.
,
Cherone
J. M.
,
Doyon
Y.
,
Ankoudinova
I.
,
Faraji
F. M.
,
Lee
A. H.
,
Ngo
C.
,
Guschin
D. Y.
,
Paschon
D. E.
,
Miller
J. C.
, et al. 
. (
2011
).
Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases.
Proc. Natl. Acad. Sci. USA
108
,
7052
7057
.

Competing interests

The authors declare no competing financial interests.

Supplementary information