Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.
Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.
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DOI：
10.1101/gr.171322.113
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发表时间：
2014-06
期刊：
影响因子：
7
通讯作者：
Kim JS
中科院分区：
文献类型：
--
作者：
Kim S;Kim D;Cho SW;Kim J;Kim JS
源自被称为CRISPR（成簇的、规律间隔的短回文重复序列）/Cas（CRISPR相关）的原核适应性免疫系统的RNA引导的工程核酸酶（RGENs）能够在人类细胞系、动物和植物中进行基因组编辑，但受到脱靶效应以及在基因组的靶位点和脱靶位点上来自编码Cas9和引导RNA的质粒的DNA片段的非预期整合的限制。在此，我们将纯化的重组Cas9蛋白和引导RNA递送到培养的人类细胞中，包括难以转染的成纤维细胞和多能干细胞。RGEN核糖核蛋白（RNPs）以高达79%的频率诱导位点特异性突变，同时减少了与在与靶位点相差一个或两个核苷酸的脱靶位点上的质粒转染相关的脱靶突变。RGEN RNPs在递送后几乎立即切割染色体DNA，并在细胞中迅速降解，从而减少脱靶效应。此外，RNP递送对人类胚胎干细胞的压力较小，产生的集落比质粒转染至少多两倍。
RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells. RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off-target mutations associated with plasmid transfection at off-target sites that differ by one or two nucleotides from on-target sites. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least twofold more colonies than does plasmid transfection.
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影响因子：
46.9
作者：
通讯作者：
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影响因子：
7
作者：
Cho SW;Kim S;Kim Y;Kweon J;Kim HS;Bae S;Kim JS
通讯作者：
Kim JS
影响因子：
46.9
作者：
Cho, Seung Woo;Kim, Sojung;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
影响因子：
7
作者：
Kim, Hye Joo;Lee, Hyung Joo;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
影响因子：
46.9
作者：
通讯作者：
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