Cas9
基因组编辑
基因组工程
亚基因组mRNA
引导RNA
计算生物学
效应器
生物
核酸酶
转录激活物样效应核酸酶
清脆的
基因组
核糖核酸
基因
遗传学
细胞生物学
作者
Prashant Mali,John Aach,P. Benjamin Stranges,Kevin M. Esvelt,Mark Moosburner,Sriram Kosuri,Luhan Yang,George M. Church
摘要
Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.
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