Department of Genetics, Stanford University, Stanford, CA, 94305, USA.
Department of Computer Science, Stanford University, Stanford, CA, 94305, USA.
Nat Commun. 2019 Sep 6;10(1):4063. doi: 10.1038/s41467-019-11955-7.
Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.
CRISPR-Cas9 基因敲除文库筛选是一种强大的方法，可用于对非编码基因组中的调控元件进行功能特征分析，但这些实验中的脱靶效应尚未得到系统评估。在这里，我们研究了 Cas9、dCas9 和 CRISPRi/a 在必需调控元件筛选实验中的脱靶活性。在 K562 细胞中进行的全基因组规模的必需 CTCF 环锚点筛选中，对基因表达影响最大的 sgRNA 并不是靠近靶标 CTCF 锚点的 sgRNA，而是具有高脱靶活性的 sgRNA，虽然与绝对脱靶位点数量的相关性较弱，但可以通过最近开发的 GuideScan 特异性评分进行预测。与不诱导双链 DNA 断裂的 CRISPRi/a 平行进行的筛选表明，一组不同的脱靶也会导致这些表观遗传编辑工具产生强烈的混杂适应度效应。有希望的是，使用 GuideScan 特异性评分对 CRISPRi 文库进行过滤，可以去除这些混杂的 sgRNA，并能够鉴定必需的调控元件。
