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Nat Commun. 2017 May 5;8:15178. doi: 10.1038/ncomms15178.

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

Author information

1
Department of Genetics, Stanford University, Stanford, California 94305, USA.
2
Department of Computer Science, Stanford University, Stanford, California 94305, USA.
3
Program in Cancer Biology, Stanford University, Stanford, California 94305, USA.
4
Stanford University Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford, California 94305, USA.

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.

PMID:
28474669
PMCID:
PMC5424143
DOI:
10.1038/ncomms15178
[Indexed for MEDLINE]
Free PMC Article

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