Figure 2. Off-target detection using nuclease-induced toxicity.

(a) Median and quartile range of full-length 19 and 20 bp guides in three cell lines by number of 1-mismatch off-targets. P values are from a Mann–Whitney test compared to guides with 0 off-targets. (b) Single mismatches closer to the PAM are less tolerated. Guides with no perfect off-targets and exactly one 1-mismatch off-targets were stratified by the location of the mismatch. Those with the location of the mismatch farther from the PAM site display greater disenrichment, demonstrating greater cutting and toxicity at these off-target sites. Error bars represent range of median results from two replicates. (c) Median and quartile range of truncated 17 and 18 bp guides in three cell lines by number of 1-mismatch off-targets. Guides with any 0-mismatch off-targets were excluded. P values are from a single-tailed Mann–Whitney test compared to guides with 0 off-targets. (d,e) Box plots of enrichment scores for guides targeting hit genes at 10% FDR for (d) growth screens and (e) a ricin screen. Signs have been flipped for ricin resistant genes for comparison. Box is length of quartile, whiskers represent 1.5 × quartile, and dots indicate enrichment of outlier guides. P values calculated using a single-tailed Mann–Whitney test. NS indicates non-significance (P>0.01).