title: Efficient genome editing of genes involved in neural crest development using the CRISPR/Cas9 system in Xenopus embryos.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, SAR, China ; KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research of Common Diseases, Hong Kong, SAR, China.
authors.forename: Zhongzhen
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Tina Tsz Kwan
authors.initials: TT
authors.lastname: Cheng
authors.affiliation: Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, South University of Science and Technology of China, Shenzhen Guangdong, 518055 China.
authors.forename: Zhaoying
authors.initials: Z
authors.lastname: Shi
authors.affiliation: Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Ziran
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Lei
authors.affiliation: Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Chengdong
authors.initials: C
authors.lastname: Wang
authors.affiliation: Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Weili
authors.initials: W
authors.lastname: Shi
authors.affiliation: Advanced Biomedical Computing Center, National Cancer Institute, National Institutes of Health, Frederick, MD 21702 USA.
authors.forename: Xiongfeng
authors.initials: X
authors.lastname: Chen
authors.affiliation: Key Laboratory for Regenerative Medicine of Ministry of Education, Ji Nan University, Guangzhou, 510632 China ; Department of Developmental and Regenerative Biology, College of Life Science and Technology, Ji Nan University, Guangzhou, 510632 China.
authors.forename: Xufeng
authors.initials: X
authors.lastname: Qi
authors.affiliation: Key Laboratory for Regenerative Medicine of Ministry of Education, Ji Nan University, Guangzhou, 510632 China ; Department of Developmental and Regenerative Biology, College of Life Science and Technology, Ji Nan University, Guangzhou, 510632 China.
authors.forename: Dongqing
authors.initials: D
authors.lastname: Cai
authors.affiliation: Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Feng
authors.affiliation: Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, South University of Science and Technology of China, Shenzhen Guangdong, 518055 China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Deng
authors.affiliation: Shenzhen Key Laboratory of Cell Microenvironment, Department of Biology, South University of Science and Technology of China, Shenzhen Guangdong, 518055 China.
authors.forename: Yonglong
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, SAR, China ; KIZ-CUHK Joint Laboratory of Bioresources and Molecular Research of Common Diseases, Hong Kong, SAR, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhao
keywords: Cas9; Gene disruption; Genome editing; Multiplex deletion; Neural crest; Segmental deletion/inversion; Xenopus