title: Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis.
abstract: 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.
authors.affiliation: Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, China.
authors.forename: Xiaogang
authors.initials: X
authors.lastname: Guo
authors.forename: Tiejun
authors.initials: T
authors.lastname: Zhang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Hu
authors.forename: Yanqi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Zhaoying
authors.initials: Z
authors.lastname: Shi
authors.forename: Qinhu
authors.initials: Q
authors.lastname: Wang
authors.forename: Yan
authors.initials: Y
authors.lastname: Cui
authors.forename: Fengqin
authors.initials: F
authors.lastname: Wang
authors.forename: Hui
authors.initials: H
authors.lastname: Zhao
authors.forename: Yonglong
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D021901:DNA, Intergenic / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism; D018628:Gene Dosage / Q000235:genetics; D018390:Gene Targeting; D005818:Genetic Engineering / Q000379:methods*; D056426:Genetic Loci / Q000235:genetics; D016678:Genome / Q000235:genetics*; D005854:Germ Cells / Q000378:metabolism; D040582:Inheritance Patterns / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D012313:RNA / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014981:Xenopus / Q000196:embryology / Q000235:genetics*
chemical_list: D021901:DNA, Intergenic; D012333:RNA, Messenger; D012313:RNA
keywords: CRISPR; Cas9; Genome editing; Xenopus tropicalis