PT - JOURNAL ARTICLE AU - Evan A Boyle AU - Winston R Becker AU - Hua B Bai AU - Janice S Chen AU - Jennifer A Doudna AU - William J Greenleaf TI - Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement AID - 10.1101/2020.09.09.290668 DP - 2020 Jan 01 TA - bioRxiv PG - 2020.09.09.290668 4099 - http://biorxiv.org/content/early/2020/09/10/2020.09.09.290668.short 4100 - http://biorxiv.org/content/early/2020/09/10/2020.09.09.290668.full AB - The RNA-guided nuclease Cas9 has unlocked powerful methods for perturbing both the genome through targeted DNA cleavage and the regulome through targeted DNA binding, but limited biochemical data has hampered efforts to quantitatively model sequence perturbation of target binding and cleavage across diverse guide sequences. We present scalable, sequencing-based platforms for high-throughput filter binding and cleavage, then perform 62,444 quantitative binding and cleavage assays on 35,047 on- and off-target DNA sequences across 90 Cas9 ribonucleoproteins (RNPs) loaded with distinct guide RNAs. We observe that binding and cleavage efficacy, as well as specificity, vary substantially across RNPs; canonically studied guides often have atypically high specificity; sequence context surrounding the target significantly influences Cas9 on-rate; and Cas9 RNPs may sequester targets in nonproductive states that contribute to “proofreading” capability. Finally, we distill our findings into an interpretable biophysical model that predicts changes in binding and cleavage for diverse target sequence perturbations.Competing Interest StatementW.J.G is a scientific co-founder of Protillion and a consultant for Guardant Health and 10x Genomics. J.S.C. is co-founder and CTO of Mammoth Biosciences. The Regents of the University of California have patents issued and pending for CRISPR technologies on which J.A.D. is an inventor. J.A.D. is a cofounder of Caribou Biosciences, Editas Medicine, Scribe Therapeutics, and Mammoth Biosciences. J.A.D. is a scientific advisory board member of Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Mammoth Biosciences, Synthego, Felix Biosciences, and Inari. J.A.D. is a Director at Johnson & Johnson and has research projects sponsored by Biogen, Pfizer, AppleTree Partners, and Roche.