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.

译文

:原核II型CRISPR-Cas系统可进行改造,以实现跨一系列真核生物的靶向基因组修饰。在这里,我们设计了这个系统,通过将转录激活结构域直接束缚至无核酸酶的Cas9蛋白或适配体修饰的单向导RNA(sgRNA),从而实现人细胞中RNA指导的基因组调控。使用此功能,我们开发了一种基于转录激活的测定法,以确定sgRNA:Cas9复合物脱靶结合的情况,并将其与转录激活剂样(TALs)效应子的脱靶活性进行了比较。我们的结果表明,特异性谱是sgRNA依赖性的,并且sgRNA:Cas9复合物和18-mer TAL效应子可以分别耐受1-3和1-2个靶错配。通过设计基因组编辑的偏移切口或通过多个协同sgRNA进行鲁棒的转录激活来设计对协同性的要求,我们提出了减轻脱靶现象的方法。我们的结果扩展了sgRNA:Cas9工具的多功能性,并强调了工程改造更高特异性的关键需求。

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