Fig. 2

CasKAS profiles sgRNA specificity genome-wide. a Summary of de novo peak calls for sgRNA #1 (using MACS2). b CasKAS signal is stronger over predicted off-target sites, but legitimate interactions are also found elsewhere in the genome. c CasKAS profile over predicted (by Cas-OFFinder) off-target sites for sgRNA #1 with dCas9 (all such sites and focusing only on the top 100 ranked by dCas9 CasKAS signal). d CasKAS profile over peak calls outside predicted (by Cas-OFFinder) off-target sites for sgRNA #1 with dCas9. e Determinants of sequence specificity as measured by dCas9 CasKAS (for sgRNA #1). PAM-distal regions of the sgRNA are less constrained than its PAM-proximal parts. The on-target sgRNA is highlighted in yellow. f Active Cas9 signal read profiles can be used to distinguish off-targets associated with cutting from those where only binding occurs. Shown are the same off-target sites as in e and the plus- and minus-strand active Cas9 5′ end profiles around the sgRNA. In this case (sgRNA #1), only the on-target site shows a Cas9 CasKAS pattern indicating cleavage; at the other sites even active Cas9 likely only binds but does not cut. A simple cutting score metric (“C-score”) based on multiplying the 5′ end forward- and reverse-strand profiles can be used to quantify cutting vs. binding. (g and h) Comparison between in vitro and in vivo CasKAS signal over predicted off-target sites for the EMX1 sgRNA. In vivo CasKAS is quantified as the difference in read per million (± 500 bp of the sgRNA site) between the sgRNA KAS-seq and the no-guide control KAS-seq (“RPM_diff”). The on-target site is shown in blue