期刊
NATURE COMMUNICATIONS
卷 8, 期 -, 页码 -出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/ncomms15178
关键词
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资金
- NIH [1DP2HD084069-01]
- NIH/NHGRI [T32 HG000044]
- Stanford ChEM-H
- Stanford Neuroscience Institute
- National Science Foundation (NSF) [DGE-114747]
- Natural Sciences and Engineering Research Council of Canada (NSERC) [PGSD3-476082-2015]
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.
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