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

Josh Tycko, Michael Wainberg, Georgi K. Marinov, Oana Ursu, Gaelen T. Hess, Braeden K. Ego, Aradhana, Amy Li, Alisa Truong, Alexandro E. Trevino, Kaitlyn Spees, David Yao, Irene M. Kaplow, Peyton G. Greenside, David W. Morgens, Douglas H. Phanstiel, Michael P. Snyder, Lacramioara Bintu, William J. Greenleaf (), Anshul Kundaje () and Michael C. Bassik ()
Additional contact information
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 G. Greenside: Stanford University
David W. Morgens: Stanford University
Douglas H. Phanstiel: Stanford University
Michael P. Snyder: Stanford University
Lacramioara Bintu: Stanford University
William J. Greenleaf: Stanford University
Anshul Kundaje: Stanford University
Michael C. Bassik: Stanford University

Nature Communications, 2019, vol. 10, issue 1, 1-14

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-11955-7

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