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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.
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. 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. 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. 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. 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. 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. S6. Cas9 and gRNA expression vectors used in this study. The upper and middle diagrams illustrate the pCS2-3xFLAG- NLS-SpCas9-NLS vector and the pUC57-Simple-gRNA backbone vector, respectively. The bottom panel shows the DNA sequence of T7 promoter, BbsI cloning sites, and the gRNA backbone. T7 promoter is underlined and the transcription start site is highlighted in yellow. The BbsI and DraI restriction sites are shaded in grey. NLS, nuclear localization signal; SpCas9, Streptococcus pyogenes Cas9.