利用CRISPR/Cas核酸酶和截短的引导RNA对人类细胞进行靶向基因组编辑。
Targeted genome editing in human cells using CRISPR/Cas nucleases and truncated guide RNAs.
作者信息
Fu Yanfang, Reyon Deepak, Joung J Keith
机构信息
Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA; Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
Molecular Pathology Unit, Center for Computational and Integrative Biology, and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA; Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
出版信息
Methods Enzymol. 2014;546:21-45. doi: 10.1016/B978-0-12-801185-0.00002-7.
CRISPR RNA-guided nucleases have recently emerged as a robust genome-editing platform that functions in a wide range of organisms. To reduce off-target effects of these nucleases, we developed and validated a modified system that uses truncated guide RNAs (tru-gRNAs). The use of tru-gRNAs leads to decreases in off-target effects and does not generally compromise the on-target efficiencies of these genome-editing nucleases. In this chapter, we describe guidelines for identifying potential tru-gRNA target sites and protocols for measuring the on-target efficiencies of CRISPR RNA-guided nucleases in human cells.
CRISPR RNA引导的核酸酶最近已成为一个强大的基因组编辑平台，可在多种生物体中发挥作用。为了减少这些核酸酶的脱靶效应，我们开发并验证了一种使用截短的引导RNA（tru-gRNA）的改良系统。tru-gRNA的使用可降低脱靶效应，并且通常不会损害这些基因组编辑核酸酶的靶向效率。在本章中，我们描述了识别潜在tru-gRNA靶位点的指南以及测量CRISPR RNA引导的核酸酶在人类细胞中的靶向效率的方案。
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