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

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, Michael P. Snyder (+3 others)
2017 Nature Communications  
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
more » ... ibrary 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.
doi:10.1038/ncomms15178 pmid:28474669 pmcid:PMC5424143 fatcat:j2l3ylfnkfhh7aprgtjqixm4pe