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Cell Biosci January 1, 2016; 6 22.

Efficient genome editing of genes involved in neural crest development using the CRISPR/Cas9 system in Xenopus embryos.

Liu Z , Cheng TT , Shi Z , Liu Z , Lei Y , Wang C , Shi W , Chen X , Qi X , Cai D , Feng B , Deng Y , Chen Y , Zhao H .

BACKGROUND: The RNA guided CRISPR/Cas9 nucleases have been proven to be effective for gene disruption in various animal models including Xenopus tropicalis. The neural crest (NC) is a transient cell population during embryonic development and contributes to a large variety of tissues. Currently, loss-of-function studies on NC development in X. tropicalis are largely based on morpholino antisense oligonucleotide. It is worthwhile establishing targeted gene knockout X. tropicails line using CRISPR/Cas9 system to study NC development. METHODS: We utilized CRISPR/Cas9 to disrupt genes that are involved in NC formation in X. tropicalis embryos. A single sgRNA and Cas9 mRNA synthesized in vitro, were co-injected into X. tropicalis embryos at one-cell stage to induce single gene disruption. We also induced duplex mutations, large segmental deletions and inversions in X. tropicalis by injecting Cas9 and a pair of sgRNAs. The specificity of CRISPR/Cas9 was assessed in X. tropicalis embryos and the Cas9 nickase was used to reduce the off-target cleavages. Finally, we crossed the G0 mosaic frogs with targeted mutations to wild type frogs and obtained the germline transmission. RESULTS: Total 16 target sites in 15 genes were targeted by CRISPR/Cas9 and resulted in successful indel mutations at 14 loci with disruption efficiencies in a range from 9.3 to 57.8 %. Furthermore, we demonstrated the feasibility of generation of duplex mutations, large segmental deletions and inversions by using Cas9 and a pair of sgRNAs. We observed that CRISPR/Cas9 displays obvious off-target effects at some loci in X. tropicalis embryos. Such off-target cleavages was reduced by using the D10A Cas9 nickase. Finally, the Cas9 induced indel mutations were efficiently passed to G1 offspring. CONCLUSION: Our study proved that CRISPR/Cas9 could mediate targeted gene mutation in X. tropicalis with high efficiency. This study expands the application of CRISPR/Cas9 platform in X. tropicalis and set a basis for studying NC development using genetic approach.

PubMed ID: 27042291
PMC ID: PMC4818404
Article link: Cell Biosci

Species referenced: Xenopus tropicalis
Genes referenced: ctnnb1 ets1 ets2 foxd3 id3 inka1 lrig3 myc myo10 pax3 plp1 snai1 snai2 sox9 tbxt.2 tfap2a twist1 tyr zic1
gRNAs referenced: ctnnb1 gRNA1 pax3 gRNA1 snai1 gRNA1 sox9 gRNA1 tfap2a gRNA1

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References [+] :
Aybar, Snail precedes slug in the genetic cascade required for the specification and migration of the Xenopus neural crest. 2002, Pubmed, Xenbase