Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells

doi:10.1038/nprot.2015.117

	Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells
	Liu, Jia1 ; Gaj, Thomas 2,3,4; Yang, Yifeng1 ; Wang, Nan1 ; Shui, Sailan1 ; Kim, Sojung 5; Kanchiswamy, Chidananda Nagamangala 6; Kim, Jin-Soo 5,7; Barbas, Carlos F., III 2,3,4
	2015-11
发表期刊	NATURE PROTOCOLS (IF:13.1[JCR-2023],17.4[5-Year])
ISSN	1754-2189
卷号	10 期号:11 页码:1842-1859
发表状态	已发表
DOI	10.1038/nprot.2015.117
摘要	Targeted nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However, realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids, the direct delivery of nuclease proteins to cells provides rapid action and fast turnover, leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression, purification and delivery of ZFN proteins, which are intrinsically cell-permeable; TALEN proteins, which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein, whose nucleofection into cells facilitates rapid induction of multiplexed modifications, are described, along with procedures for evaluating nuclease protein activity. Once they are constructed, nuclease proteins can be expressed and purified within 6 d, and they can be used to induce genomic modifications in human cells within 2 d.
收录类别	SCI
语种	英语
资助项目	Institute for Basic Science[IBS-R021-D1]
WOS研究方向	Biochemistry & Molecular Biology
WOS类目	Biochemical Research Methods
WOS记录号	WOS:000363472700012
出版者	NATURE PUBLISHING GROUP
WOS关键词	ZINC-FINGER-NUCLEASE ; TARGETED GENE KNOCKOUT ; IN-VIVO ; HIGHLY EFFICIENT ; MAMMALIAN-CELLS ; LENTIVIRAL VECTORS ; MEDIATED DELIVERY ; CRYSTAL-STRUCTURE ; DNA RECOGNITION ; RNA
原始文献类型	Article
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/2103
专题	生命科学与技术学院免疫化学研究所_特聘教授组_抗体化学实验室免疫化学研究所_特聘教授组_功能筛选实验室生命科学与技术学院_硕士生免疫化学研究所_PI研究组_刘佳组
通讯作者	Liu, Jia
作者单位	1.ShanghaiTech Univ, Shanghai Inst Adv Immunochem Studies, Shanghai, Peoples R China 2.Scripps Res Inst, Skaggs Inst Chem Biol, La Jolla, CA 92037 USA 3.Scripps Res Inst, Dept Chem, La Jolla, CA 92037 USA 4.Scripps Res Inst, Dept Cell & Mol Biol, La Jolla, CA 92037 USA 5.Inst for Basic Sci Korea, Ctr Genome Engn, Seoul, South Korea 6.FEM, Res & Innovat Ctr, Dept Genom & Biol Fruit Crop, San Michele All Adige, Italy 7.Seoul Natl Univ, Dept Chem, Seoul, South Korea
第一作者单位	免疫化学研究所
通讯作者单位	免疫化学研究所
第一作者的第一单位	免疫化学研究所
推荐引用方式 GB/T 7714	Liu, Jia,Gaj, Thomas,Yang, Yifeng,et al. Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells[J]. NATURE PROTOCOLS,2015,10(11):1842-1859.
APA	Liu, Jia.,Gaj, Thomas.,Yang, Yifeng.,Wang, Nan.,Shui, Sailan.,...&Barbas, Carlos F., III.(2015).Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells.NATURE PROTOCOLS,10(11),1842-1859.
MLA	Liu, Jia,et al."Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells".NATURE PROTOCOLS 10.11(2015):1842-1859.