CRISPR/Cas(clustered regularly interspaced short palindromic repeats/CRISPR-associated system)存在于大多数的细菌和几乎所有的古细菌中,是细菌在进化过程中形成的一种免疫防御机制,以帮助细菌抵御外来病毒和 DNA 的入侵。其中 Cas9(CRISPR-associated protein 9)具有用于基因操作的潜在可能。 原理 Cas9 是一种内切酶,其...
通过将表达sgRNA的原件与表达Cas9的原件相连接,得到可以同时表达两者的质粒,将其转染细胞,便能够对目的基因进行基因操作,在PAM (5’-NGG) 上游 ~3bp进行切割,如下图所示。 因此本实验的关键步骤是设计一对20bp(不包括NGG在内的)完全互补的oligo...
一CRISPR-Cas系统简介 图1 CRISPR-Cas9系统介绍 CRISPR-Cas9 系统是一种被广泛运用的基因组编辑工具,它来源于细菌的适应性免 疫系统。CRISPR-Cas9系统包括:Cas9酶和一个向导RNA。 向导RNA作用是引导 cas9 到基因组的特异性位点上切割。如图1所示。目前为止,CRISPR-Cas9 系统主 要有两方面应用: 基因敲除和基因敲入...
12、.2. Wang H, Yang H, Sliivalila CS, Dawlaty MM, Cheng AW, Zliang F, and Jaeiiisch R Onestep generation of mice canying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910-&3. Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, Yan ...
sgRNA,单链引导 RNA,作为 CRISPR/Cas9 系统中 cas 蛋白的引导序列,可参照 sgRNA 工具网站(http://crispr.mit.edu)设计多条 sgRNA 以降低脱靶率。 sgRNA 质粒的构建方法 1)用 BbsI 在 37℃条件下酶切载体质粒 pAC1371。 2)琼脂糖凝胶电泳,切胶用 DNA 片段回收试剂盒回收目的载体片段 ...
CRISPR/Cas9: structure, mechanisms of action, efficacy, off-target activity, genetic backgrounds, multi-targeting, point-mutations, and large deletions.CRISPR/Cas9 structure and mechanisms of action A CRISPR/Cas9 nuclease system requires two components: a Cas enzyme for cutting the target sequence and...
先看中文文章可以快速了解背景,而后根据实验设计需求,钻研好的英文protocol。 目前已有的基因编辑技术 (针对这点应该加以展开,不过这里我只对自己需要用的CRISPR-Cas9进行了解) (1)Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 第三代人 ...
The rise of the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein (Cas) system has made it possible to induce double-strand breaks at almost any desired target site in the genome. In plant somatic cells, double-strand breaks are predominantly repaired by ...
(TALENs); and the most widely used third generation gene-editing technology is clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas).4Unlike ZFNs and TALENs, which use proteins to target DNA strands, CRISPR technology directs Cas proteins to a ...
No new data were generated or analyzed with this protocol; all presented data were previously published9,15. References Watve, M. G., Tickoo, R., Jog, M. M. & Bhole, B. D. How many antibiotics are produced by the genus Streptomyces? Arch. Microbiol. 176, 386–390 (2001). CAS Pu...