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

Josh Tycko(Stanford University), Michael Wainberg(Stanford University), Georgi K. Marinov(Stanford University), Oana Ursu(Stanford University), Gaelen T. Hess(Stanford University), Braeden K. Ego(Stanford University), Aradhana(Stanford University), Amy Li(Stanford University), Alisa Truong(Stanford University), Alexandro E. Trevino(Stanford University), Kaitlyn Spees(Stanford University), David Yao(Stanford University), Irene M. Kaplow(Stanford University), Peyton Greenside(Stanford University), David W. Morgens(Stanford University), Douglas H. Phanstiel(University of North Carolina at Chapel Hill), M Snyder(Stanford University), Lacramioara Bintu(Stanford University), William J. Greenleaf(Chan Zuckerberg Initiative (United States)), Anshul Kundaje(Stanford Medicine), Michael C. Bassik(Stanford Health Care)
Nature Communications
September 6, 2019
10.1038/s41467-019-11955-7
Cited by 166Open Access
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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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