GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos

doi:10.1038/s41596-020-0361-1

	GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos
	Zuo, Erwei 1,2; Sun, Yidi 1,3; Wei, Wu 4,5,6; Yuan, Tanglong 2; Ying, Wenqin 1; Sun, Hao 7; Yuan, Liyun 4; Steinmetz, Lars M.6,8,9; Li, Yixue4,10,11,12,13 ; Yang, Hui 1
	2020-08-14
发表期刊	NATURE PROTOCOLS (IF:13.1[JCR-2023],17.4[5-Year])
ISSN	1754-2189
EISSN	1750-2799
发表状态	已发表
DOI	10.1038/s41596-020-0361-1
摘要	Genome editing holds great potential for correcting pathogenic mutations. We developed a method called GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. GOTI directly compares edited and non-edited cells without the interference of genetic background and thus could detect potential off-target variants with high sensitivity. Notably, the GOTI method was designed to detect potential off-target variants of any genome editing tools by the combination of experimental and computational approaches, which is critical for accurate evaluation of the safety of genome editing tools. Here we provide a detailed protocol for GOTI, including mice mating, two-cell embryo injection, embryonic day 14.5 embryo digestion, fluorescence-activated cell sorting, whole-genome sequencing and data analysis. To enhance the utility of GOTI, we also include a computational workflow called GOTI-seq (https://github.com/sydaileen/GOTI-seq) for the sequencing data analysis, which can generate the final genome-wide off-target variants from raw sequencing data directly. The protocol typically takes 20 d from the mice mating to sequencing and 7 d for sequencing data analysis.
收录类别	SCI ; SCIE
语种	英语
资助项目	R&D Program of China[2018YFC2000100][2017YFC1001302][2017YFC0908405] ; CAS Strategic Priority Research Program[XDB32060000] ; National Natural Science Foundation of China[31871502][31522037] ; Shanghai Municipal Science and Technology Major Project[2018SHZDZX05] ; Shanghai City Committee of Science and Technology Project[18411953700][18JC1410100] ; National Institutes of Health P01 Center grant[P01HG00020527]
WOS研究方向	Biochemistry & Molecular Biology
WOS类目	Biochemical Research Methods
WOS记录号	WOS:000559668000001
出版者	NATURE PUBLISHING GROUP
WOS关键词	CRISPR-CAS9 ; DNA ; BASE ; SEQ ; MUTATIONS ; CLEAVAGE ; REVEALS ; FUTURE
原始文献类型	Article ; Early Access
引用统计	正在获取...
文献类型	期刊论文
条目标识符	https://kms.shanghaitech.edu.cn/handle/2MSLDSTB/122911
专题	生命科学与技术学院_特聘教授组_李亦学组
通讯作者	Steinmetz, Lars M.; Li, Yixue; Yang, Hui
作者单位	1.Chinese Acad Sci, CAS Ctr Excellence Brain Sci & Intelligence Techn, Shanghai Inst Biol Sci,Shanghai Res Ctr Brain Sci, Inst Neurosci,State Key Lab Neurosci,Key Lab Prim, Shanghai, Peoples R China 2.Chinese Acad Agr Sci, Agr Genom Inst Shenzhen, Shenzhen Branch,Minist Agr, Guangdong Lab Lingnan Modern Agr,Genome Anal Lab, Shenzhen, Peoples R China 3.Chinese Acad Sci, Univ Chinese Acad Sci, CAS Ctr Excellence Mol Cell Sci, Inst Biochem & Cell Biol,CAS Key Lab Syst Biol,Sh, Shanghai, Peoples R China 4.Univ Chinese Acad Sci, Chinese Acad Sci Shanghai, Biomed Big Data Ctr,Shanghai Inst Nutr & Hlth,Sha, CAS MPG Partner Inst Computat Biol,Key Lab Comput, Shanghai, Peoples R China 5.Shanghai Jiao Tong Univ, Shanghai Childrens Hosp, Ctr Biomed Informat, Shanghai, Peoples R China 6.Stanford Univ, Stanford Genome Technol Ctr, Palo Alto, CA 94304 USA 7.Univ Calif San Diego, La Jolla, CA 92093 USA 8.Stanford Univ, Sch Med, Dept Genet, Stanford, CA 94305 USA 9.European Mol Biol Lab EMBL, Genome Biol Unit, Heidelberg, Germany 10.Shanghai Tech Univ, Dept Life Sci, Shanghai, Peoples R China 11.Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, Shanghai, Peoples R China 12.Fudan Univ, Collaborat Innovat Ctr Genet & Dev, Shanghai, Peoples R China 13.Shanghai Acad Sci & Technol, Shanghai Ctr Bioinformat Technol, Shanghai, Peoples R China
通讯作者单位	上海科技大学
推荐引用方式 GB/T 7714	Zuo, Erwei,Sun, Yidi,Wei, Wu,et al. GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos[J]. NATURE PROTOCOLS,2020.
APA	Zuo, Erwei.,Sun, Yidi.,Wei, Wu.,Yuan, Tanglong.,Ying, Wenqin.,...&Yang, Hui.(2020).GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos.NATURE PROTOCOLS.
MLA	Zuo, Erwei,et al."GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos".NATURE PROTOCOLS (2020).