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.

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Xenopus tropicalis is an emerging vertebrate genetic model. A gene knock-in method has not yet been reported in this species. Here, we report that heritable targeted integration can be achieved in this diploid frog using a concurrent cleavage strategy mediated by the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/ Cas9) system. The key point of the strategy is the addition of a Cas9/guide RNA cleavage site in the donor vector, allowing simultaneous cutting of the chromosomal target site and circular donor DNA in vivo. For the 3 distinct loci tested, all showed efficient targeted integration that was verified by both germ-line transmission and Southern blot analyses. By designing the target sites in introns, we were able to get precise editing of the tyrosinase coding sequence and green fluorescent protein expression from endogenous n-tubulin promoter and enhancers. We were unable to detect off-target effects with the T7 endonuclease I assay. Precise editing of protein coding sequences in X. tropicalis expands the utility of this diploid frog, such as for establishing models to study human inherited diseases.-Shi, Z., Wang, F., Cui, Y., Liu, Z., Guo, X., Zhang, Y., Deng, Y., Zhao, H., Chen, Y. Heritable CRISPR/Cas9-mediated targeted integration in Xenopus tropicalis. FASEB J. 29, 4914-4923 (2015). www.fasebj.orgRecent establishment of efficient targeted gene disruption methods in Xenopus tropicalis (1-6) has confirmed this diploid frog as an excellent vertebrate genetic model; however, recombination-mediated genome editing has not been reported in this species.Direct injection of mRNAs of engineered nucleases, such as zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), into fertilized eggs facilitates homologybased integration in zebrafish, mouse, and rat embryos (7-11). Unfortunately, taking similar strategies with either circular donor DNA, linear donor DNA, or synthetic oligodeoxynucleotides, we were unable to get heritable homologous recombination-mediated gene modification in X. tropicalis in the past several years, likely as a result of too low recombination efficiency.Here, we show that heritable targeted integration in X. tropicalis was successfully obtained when we took a homology-independent strategy recently developed for cell lines and zebrafish (12-15). The most prominent feature of the strategy is the introduction of a nuclease cleavage site into the donor DNA, thus allowing the concurrent cleavage of the chromosomal target site and circular donor DNA in vivo by a given nuclease. In addition, we designed a reporter system that helps identify early integration events. We tested the editing of 3 genes in X. tropicalis with this strategy. All showed efficient targeted integration that was passed to the next generation through germ-line transmission and was further confirmed by Southern blot analysis. Our data r...