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Development 2014 Feb 01;1413:707-14. doi: 10.1242/dev.099853.
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Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis.

Guo X , Zhang T , Hu Z , Zhang Y , Shi Z , Wang Q , Cui Y , Wang F , Zhao H , Chen Y .

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.

PubMed ID: 24401372
Article link: Development

Species referenced: Xenopus tropicalis
Genes referenced: cela1 cela1.2 cela1.6 ets1 ets2 hhex hspa5 pam pdia2 pdx1 pgat ptf1a tm4sf4 tyr
GO keywords: pancreas development
gRNAs referenced: cela1.6 gRNA1 cela1.6 gRNA2 ets1 gRNA1 ets1 gRNA2 ets1 gRNA3 ets2 gRNA1 hhex gRNA1 pdx1 gRNA1 pdx1 gRNA2 pdx1 gRNA3 pgat gRNA1 pgat gRNA2 pgat gRNA3 ptf1a gRNA1 tm4sf4 gRNA1 tm4sf4 gRNA2 tyr gRNA12

Phenotypes: Xtr Wt + hhex CRISPR (Fig. 1 ABCD) [+]

Article Images: [+] show captions