Efficient Generation of Gene-Modified Pigs Harboring Precise Orthologous Human Mutation via CRISPR/Cas9-Induced Homology-Directed Repair in Zygotes
2016-01
Source PublicationHUMAN MUTATION
ISSN1059-7794
Volume37Issue:1Pages:110-118
Status已发表
DOI10.1002/humu.22913
Abstract

Precise genetic mutation of model animals is highly valuable for functional investigation of human mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-induced homology-directed repair (HDR) is usually used for precise genetic mutation, being limited by the relatively low efficiency compared with that of non-homologous end joining (NHEJ). Although inhibition of NHEJ was shown to enhance HDR-derived mutation, in this work, without inhibition of NHEJ, we first generated gene-modified pigs harboring precise orthologous human mutation (Sox10 c.A325>T) via CRISPR/Cas9-induced HDR in zygotes using single-strand oligo DNA (ssODN) as template with an efficiency as high as 80%, indicating that pig zygotes exhibited high activities of HDR relative to NHEJ and were highly amendable to genetic mutation via CIRSPR/Cas9-induced HDR. Besides, we found a higher concentration of ssODN remarkably reduced HDR-derived mutation in pig zygotes, suggesting a possible balance for optimal HDR-derived mutation in zygotes between the excessive accessibility to HDR templates and the activities of HDR relative to NHEJ which appeared to be negatively correlated to ssODN concentration. In addition, the HDR-derived mutation, as well as those from NHEJ, extensively integrated into various tissues including gonad of founder pig without detected off-targeting, suggesting CRISPR/Cas9-induced HDR in zygotes is a reliable approach for precise genetic mutation in pigs.

KeywordSOX10 CRISPR Cas9 homology-directed repair pigs model organism disease model
Indexed BySCI
Language英语
Funding ProjectNational Major Scientific Projects Specifically for R&D of Transgenic Organisms[2014ZX08009-003-006]
WOS Research AreaGenetics & Heredity
WOS SubjectGenetics & Heredity
WOS IDWOS:000369159900011
PublisherWILEY
WOS KeywordCONDITIONAL ALLELES ; CLONING EFFICIENCY ; KNOCKOUT RATS ; NEURAL CREST ; SOX10 ; SPECIFICITY ; CLEAVAGE
Original Document TypeArticle
Citation statistics
Document Type期刊论文
Identifierhttps://kms.shanghaitech.edu.cn/handle/2MSLDSTB/2053
Collection生命科学与技术学院_PI研究组_黄行许组
Corresponding AuthorWang, Yong; Wei, Hong
Affiliation
1.Third Mil Med Univ, Coll Basic Med Sci, Dept Lab Anim Sci, Chongqing, Peoples R China
2.ShanghaiTech Univ, Sch Life Sci & Technol, Shanghai, Peoples R China
Recommended Citation
GB/T 7714
Zhou, Xiaoyang,Wang, Lulu,Du, Yinan,et al. Efficient Generation of Gene-Modified Pigs Harboring Precise Orthologous Human Mutation via CRISPR/Cas9-Induced Homology-Directed Repair in Zygotes[J]. HUMAN MUTATION,2016,37(1):110-118.
APA Zhou, Xiaoyang.,Wang, Lulu.,Du, Yinan.,Xie, Fei.,Li, Liang.,...&Wei, Hong.(2016).Efficient Generation of Gene-Modified Pigs Harboring Precise Orthologous Human Mutation via CRISPR/Cas9-Induced Homology-Directed Repair in Zygotes.HUMAN MUTATION,37(1),110-118.
MLA Zhou, Xiaoyang,et al."Efficient Generation of Gene-Modified Pigs Harboring Precise Orthologous Human Mutation via CRISPR/Cas9-Induced Homology-Directed Repair in Zygotes".HUMAN MUTATION 37.1(2016):110-118.
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