Systematic profiling of in vitro binding affinity and endonuclease activity of CRISPR enzymes

CRISPR RNA-guided endonucleases (RGEs) are common tools for genomic editing and directing activities to genomic loci, yet each has off-target effects that are unpredictable. We developed a pair of simple assays to systematically measure the DNA-binding specificity (Spec-seq), catalytic activity specificity (SEAM-seq), and cleavage efficiency of RGEs and generate feature-based models. The models for SpCas9 were consistent with previous reports of its in vitro and in vivo specificity, validating the approach. Further, by disentangling binding from cleavage, Spec/SEAM-seq provides mechanistic insight into off-target activity by revealing target-dependent differences in DNA-binding specificity and a kinetic phenomenon we term "mismatch activation". Spec/SEAM-seq is also useful for profiling less-well characterized RGEs. Application to an engineered SpCas9, HiFi-SpCas9, indicated that its enhanced target discrimination can be attributed to cleavage rather than binding specificity. The ortholog ScCas9, on the other hand, derives specificity from binding to an extended PAM. The decreased off-target activity of AsCas12a (Cpf1) appears to be primarily driven by DNA-binding specificity. Finally, we performed the first characterization of CasX specificity, revealing an all-or-nothing mechanism where mismatches can be bound, but not cleaved. These applications establish Spec/SEAM-seq as an accessible method to rapidly and reliably evaluate the specificity of RGEs and Cas::gRNA pairs and provide insight into the mechanism and thermodynamics of target discrimination.