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PMID: 28474669 Published · epublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens.

Nature communications ·Vol. 8 ·2017-00-05 ·Pages 15178

Morgens DW, Wainberg M, Boyle EA, Ursu O, Araya CL, Tsui CK, Haney MS, Hess GT, Han K, Jeng EE, Li A, Snyder MP, Greenleaf WJ, Kundaje A, Bassik MC

Abstract

CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.

MeSH Terms
CRISPR-Cas Systems/genetics Cell Line Clustered Regularly Interspaced Short Palindromic Repeats/genetics DNA Damage/genetics Gene Targeting/methods Genomic Library High-Throughput Screening Assays/methods Humans Polysaccharides/biosynthesis RNA Interference RNA, Guide/genetics Ricin/toxicity
Chemicals
Polysaccharides RNA, Guide Ricin
Authors & Affiliations
15 authors, click to expand affiliations / ORCID
Morgens David W
Department of Genetics, Stanford University, Stanford, California 94305, USA.
Wainberg Michael
Department of Computer Science, Stanford University, Stanford, California 94305, USA.
Boyle Evan A ORCID
Department of Genetics, Stanford University, Stanford, California 94305, USA.
Ursu Oana
Department of Genetics, Stanford University, Stanford, California 94305, USA.
Araya Carlos L
Department of Genetics, Stanford University, Stanford, California 94305, USA.
Tsui C Kimberly
Department of Genetics, Stanford University, Stanford, California 94305, USA.
Haney Michael S
Department of Genetics, Stanford University, Stanford, California 94305, USA.
Hess Gaelen T
Department of Genetics, Stanford University, Stanford, California 94305, USA.
Han Kyuho ORCID
Department of Genetics, Stanford University, Stanford, California 94305, USA.
Jeng Edwin E
Department of Genetics, Stanford University, Stanford, California 94305, USA. | Program in Cancer Biology, Stanford University, Stanford, California 94305, USA.
Li Amy
Department of Genetics, Stanford University, Stanford, California 94305, USA.
Snyder Michael P
Department of Genetics, Stanford University, Stanford, California 94305, USA.
Greenleaf William J
Department of Genetics, Stanford University, Stanford, California 94305, USA.
Kundaje Anshul ORCID
Department of Genetics, Stanford University, Stanford, California 94305, USA. | Department of Computer Science, Stanford University, Stanford, California 94305, USA.
Bassik Michael C
Department of Genetics, Stanford University, Stanford, California 94305, USA. | Stanford University Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford, California 94305, USA.
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Article Info
Journal
Nature communications
Abbr.
Nat Commun
ISSN
2041-1723
DOI
10.1038/ncomms15178
Published
2017-00-05
Epub
2017-00-05
Pages
15178
Language
English
Region
England
NLM ID
101528555
PMCID
PMC5424143
Subset
IM
Grants
NHGRI NIH HHS · P50 HG007735 · United States
NIGMS NIH HHS · T32 GM007790 · United States
NHGRI NIH HHS · T32 HG000044 · United States
External Links
PubMed source DOI: 10.1038/ncomms15178 PMC Full Text View journal details
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