Cas9
清脆的
引导RNA
基因组编辑
转录激活物样效应核酸酶
回文
核糖核酸
计算生物学
生物
基因组工程
遗传学
基因
作者
D. Dewran Koçak,Eric A. Josephs,Vidit Bhandarkar,Shaunak Adkar,Jennifer B. Kwon,Charles A. Gersbach
标识
DOI:10.1038/s41587-019-0095-1
摘要
CRISPR (clustered regularly interspaced short palindromic repeat) systems have been broadly adopted for basic science, biotechnology, and gene and cell therapy. In some cases, these bacterial nucleases have demonstrated off-target activity. This creates a potential hazard for therapeutic applications and could confound results in biological research. Therefore, improving the precision of these nucleases is of broad interest. Here we show that engineering a hairpin secondary structure onto the spacer region of single guide RNAs (hp-sgRNAs) can increase specificity by several orders of magnitude when combined with various CRISPR effectors. We first demonstrate that designed hp-sgRNAs can tune the activity of a transactivator based on Cas9 from Streptococcus pyogenes (SpCas9). We then show that hp-sgRNAs increase the specificity of gene editing using five different Cas9 or Cas12a variants. Our results demonstrate that RNA secondary structure is a fundamental parameter that can tune the activity of diverse CRISPR systems. Changes to the secondary structure of the guide RNA enable substantial increases in specificity of Cas9 and Cas12 variants
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