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; Michael C Bassik

doi:10.1038/s41467-019-11955-7

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

Nat Commun. 2019 Sep 6;10(1):4063. doi: 10.1038/s41467-019-11955-7.

Authors

Josh Tycko¹, Michael Wainberg², Georgi K Marinov¹, Oana Ursu¹, Gaelen T Hess¹, Braeden K Ego¹, Aradhana¹, Amy Li¹, Alisa Truong¹, Alexandro E Trevino^{3

4}, Kaitlyn Spees¹, David Yao¹, Irene M Kaplow^{2

5}, Peyton G Greenside^{1

6}, David W Morgens¹, Douglas H Phanstiel^{1

7

8}, Michael P Snyder¹, Lacramioara Bintu⁴, William J Greenleaf^{9

10

11}, Anshul Kundaje^{12

13}, Michael C Bassik^{14

15}

Affiliations

¹ Department of Genetics, Stanford University, Stanford, CA, 94305, USA.
² Department of Computer Science, Stanford University, Stanford, CA, 94305, USA.
³ Center for Personal Dynamic Regulomes, Stanford University, Stanford, CA, 94305, USA.
⁴ Department of Bioengineering, Stanford University, Stanford, CA, 94305, USA.
⁵ Department of Biology, Stanford University, Stanford, CA, 94305, USA.
⁶ Program in Biomedical Informatics, Stanford University School of Medicine, Stanford, CA, 94305, USA.
⁷ Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC, 27599, USA.
⁸ Thurston Arthritis Research Center, University of North Carolina, Chapel Hill, NC, 27599, USA.
⁹ Department of Genetics, Stanford University, Stanford, CA, 94305, USA. wjg@stanford.edu.
¹⁰ Department of Applied Physics, Stanford University, Stanford, CA, 94305, USA. wjg@stanford.edu.
¹¹ Chan Zuckerberg Biohub, San Francisco, CA, 94158, USA. wjg@stanford.edu.
¹² Department of Genetics, Stanford University, Stanford, CA, 94305, USA. akundaje@stanford.edu.
¹³ Department of Computer Science, Stanford University, Stanford, CA, 94305, USA. akundaje@stanford.edu.
¹⁴ Department of Genetics, Stanford University, Stanford, CA, 94305, USA. bassik@stanford.edu.
¹⁵ Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford University, Stanford, CA, 94305, USA. bassik@stanford.edu.

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.

Publication types

Research Support, N.I.H., Extramural

MeSH terms

CRISPR-Cas Systems*
Computational Biology / methods
Epigenesis, Genetic / genetics
Epigenomics / methods
Gene Editing / methods
Gene Expression Regulation, Neoplastic*
Genome, Human / genetics*
HEK293 Cells
Humans
K562 Cells
RNA, Guide, CRISPR-Cas Systems / genetics*
Regulatory Elements, Transcriptional / genetics*

Substances

RNA, Guide, CRISPR-Cas Systems

Abstract

Publication types

MeSH terms

Substances

Grants and funding