Efficient genome editing of genes involved in neural crest development using the CRISPR/Cas9 system in Xenopus embryos. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Liu, Zhongzhen; Cheng, Tina; Shi, Zhaoying; Liu, Ziran; Lei, Yong; Wang, Chengdong; Shi, Weili; Chen, Xiongfeng; Qi, Xufeng; Cai, Dongqing; Feng, Bo; Deng, Yi; Chen, Yonglong; Zhao, Hui
• Is Part Of: Cell & bioscience. Volume 6:Issue 1(2016)
• Page Start: 1
• Page End: 13
• Publication Date: 2016-12
• Subjects: Genome editing -- Cas9 -- Neural crest -- Gene disruption -- Segmental deletion/inversion -- Multiplex deletion -- Xenopus; Biology -- Periodicals; Life sciences -- Periodicals; Cytology -- Periodicals; 571.6
• DOI: 10.1186/s13578-016-0088-4
• Languages: English
• ISSNs: 2045-3701

Abstract

Abstract Background The RNA guided CRISPR/Cas9 nucleases have been proven to be effective for gene disruption in various animal models includingXenopus 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 inX. tropicalis are largely based on morpholino antisense oligonucleotide. It is worthwhile establishing targeted gene knockoutX. tropicails line using CRISPR/Cas9 system to study NC development. Methods We utilized CRISPR/Cas9 to disrupt genes that are involved in NC formation inX. tropicalis embryos. A single sgRNA andCas9 mRNA synthesized in vitro, were co-injected intoX. tropicalis embryos at one-cell stage to induce single gene disruption. We also induced duplex mutations, large segmental deletions and inversions inX. tropicalis by injectingCas9 and a pair of sgRNAs. The specificity of CRISPR/Cas9 was assessed inX. 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 inX. 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 inX. tropicalis with high efficiency. This study expands the application of CRISPR/Cas9 platform inX. tropicalis and set a basis for studying NC development using genetic approach.