Mitigation of off-target toxicity in CRISPR-Cas9 screens for essential non-coding elements.

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ID: 40193
2019
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Abstract
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.
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tycko2019mitigationnature Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Tycko, Josh;Wainberg, Michael;Marinov, Georgi K;Ursu, Oana;Hess, Gaelen T;Ego, Braeden K;Aradhana, ;Li, Amy;Truong, Alisa;Trevino, Alexandro E;Spees, Kaitlyn;Yao, David;Kaplow, Irene M;Greenside, Peyton G;Morgens, David W;Phanstiel, Douglas H;Snyder, Michael P;Bintu, Lacramioara;Greenleaf, William J;Kundaje, Anshul;Bassik, Michael C;
Journal Nature communications
Year 2019
DOI
10.1038/s41467-019-11955-7
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