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

2017; Nature Portfolio; Volume: 8; Issue: 1 Linguagem: Inglês

10.1038/ncomms15178

2041-1723

David W. Morgens, Michael Wainberg, Evan A. Boyle, Oana Ursu, Carlos L. Araya, C. Kimberly Tsui, Michael S. Haney, Gaelen T. Hess, Kyuho Han, Edwin E. Jeng, Amy Li, M Snyder, William J. Greenleaf, Anshul Kundaje, Michael C. Bassik,

RNA and protein synthesis mechanisms

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.