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

Tycko J, Wainberg M, Marinov GK, Ursu O, Hess GT, Ego BK, Aradhana, Li A, Truong A, Trevino AE, Spees K, Yao D, Kaplow IM, Greenside PG, Morgens DW, Phanstiel DH, Snyder MP, Bintu L, Greenleaf WJ, Kundaje A, Bassik MC.
Nature communications. 2019 Sep 6;10(1):4063.
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.