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

  • Author(s): 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
  • et al.
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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