Self-Cloning CRISPR.
Self-Cloning CRISPR.
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DOI：
10.1002/cpsc.14
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发表时间：
2016-08-17
影响因子：
--
通讯作者：
Sherwood, Richard I
中科院分区：
文献类型：
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作者：
Arbab, Mandana;Sherwood, Richard I
CRISPR/Cas9-基因编辑已经成为一项革命性的技术，通过设计互补的sgRNA序列并将其与Cas9一起引入细胞中，可以轻松地修改特定的基因组座位。自克隆CRISPR/Cas9(ScCRISPR)利用自切割回文sgRNA质粒(SgPal)，通过同源重组在靶细胞内与短的PCR扩增的位点特异的sgRNA序列重组，绕过sgRNA质粒的构建过程。通过这种机制，一旦sgRNA寡核苷酸可用，scCRISPR就可以在2小时内进行基因编辑，其效率相当于传统的sgRNA靶向：>；在小鼠和人类胚胎干细胞和癌细胞系中90%的基因敲除。此外，使用基于PCR的短同源臂添加，我们实现了对GFP等转基因基因的高效定点敲入，而不需要传统的质粒克隆或基因组整合选择盒(敲入率为2%~4%)。本文介绍了CRISPR基因编辑最快速、最有效的方法。2016John Wiley&Sons，Inc.
CRISPR/Cas9-gene editing has emerged as a revolutionary technology to easily modify specific genomic loci by designing complementary sgRNA sequences and introducing these into cells along with Cas9. Self-cloning CRISPR/Cas9 (scCRISPR) uses a self-cleaving palindromic sgRNA plasmid (sgPal) that recombines with short PCR-amplified site-specific sgRNA sequences within the target cell by homologous recombination to circumvent the process of sgRNA plasmid construction. Through this mechanism, scCRISPR enables gene editing within 2 hr once sgRNA oligos are available, with high efficiency equivalent to conventional sgRNA targeting: >90% gene knockout in both mouse and human embryonic stem cells and cancer cell lines. Furthermore, using PCR-based addition of short homology arms, we achieve efficient site-specific knock-in of transgenes such as GFP without traditional plasmid cloning or genome-integrated selection cassette (2% to 4% knock-in rate). The methods in this paper describe the most rapid and efficient means of CRISPR gene editing. © 2016 by John Wiley & Sons, Inc.