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

The CRISPR family of RNA-guided endonucleases have become a common tool for genomic editing and directing other activities to specific genomic loci. However, the most widely adopted enzyme, SpCas9, exhibits substantial and poorly understood off-target binding and cleavage that restricts its clinical and research use. In this work we show that a pair of simple and quick assays enable systematic evaluation of DNA-binding specificity (Spec-seq), catalytic activity (SEAM-seq), and overall cleavage efficiency for off-target sequences. Using feature-based models fit to these data, we show that the in vitro cleavage efficiency of Cas9 is consistent with in vivo activity as measured by the Doench rules. We also find that Cas9 binding specificity and cleavage activity differ dramatically between the target sequences. This includes mismatch activation, a kinetic phenomenon in which cleavage of off-target sites are cleaved more slowly, but more completely, than on-target sites. Applying the same approach, we observed that AsCas12a (aka AsCpf1) cleavage specificity is highly dependent on the DNA binding specificity and in insensitive to the composition of the target sequence. In summary, our methodology can be used to rapidly and reliably evaluate the targeting and editing of Cas:gRNA pairs and new CRISPR/Cas variants and lend insight into the mechanism and thermodynamics of target discrimination.