Abstract
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 have 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 and 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 modulates Cas9 on-rate; and Cas9 RNPs may sequester targets in nonproductive states that contribute to "proofreading" capability. Lastly, we distill our findings into an interpretable biophysical model that predicts changes in binding and cleavage for diverse target sequence perturbations.
Highlights
Streptococcus pyogenes (Spy) Cas9 has been widely adopted as a platform for perturbing gene expression and protein levels in human cells [1]
The native CRISPR-Cas9 bacterial system has been engineered to bind to DNA without inducing cleavage as a catalytically inactive Cas9. dCas9 has proven a powerful platform for modulating gene expression, when fused to effector domains to permit perturbation of specific genomic loci [2]
Such work has revealed that the Cas9 ribonucleoprotein (RNP) first associates to an NGG protospacer adjacent motif (PAM) and hybridizes to 8 to 12 target nucleotides abutting the PAM known as the “seed” region
Summary
Streptococcus pyogenes (Spy) Cas has been widely adopted as a platform for perturbing gene expression and protein levels in human cells [1] In this type II CRISPR system, the CRISPR-associated protein Cas performs targeted search and cleavage of double-stranded DNA guided by a CRISPR RNA (crRNA) that is complementary to the target sequence. Such work has revealed that the Cas ribonucleoprotein (RNP) first associates to an NGG protospacer adjacent motif (PAM) and hybridizes to 8 to 12 target nucleotides abutting the PAM known as the “seed” region. Mismatches within this seed region inhibit stable RNP:target complex formation, whereas mismatches. We develop a predictive biophysical model for Cas binding and cleavage of off-target sites
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