Fig. 2: Off-target activity levels estimates by CRISPECTOR.

a Site 51 of RAG1, XT 2p in HEK293-Cas9. High rate of deletions of length 1 at the expected cut-site masks the real editing signal (Tx=17, M = 41, solid circle) if only subtraction is used. Also, the PAM site is GGG. Thus, we can find an alternative cut-site 1-bp to the right of the expected cut-site. CRISPECTOR points to five reads with deletions of length 1 on this alternative cut-site (dashed circle). Editing activity estimation—CRISPECTOR = 0.113%, CRISPResso2 = 0.068% and ampliCan = 0.070%. Note that reference is depicted 3’→5’ since the gRNA cuts the negative strand in this locus. The notation Del k stands for deletions of length k. b Editing activity by CRISPECTOR, ampliCan and CRISPResso2 for RAG1, XT 2p, HEK293-Cas9 sample. For many instances with low editing activity levels, there is a disagreement between tools with respect to the active (≥0.1%) or inactive (<0.1%) classification. In this experiment, CRISPECTOR reported four sites as ≥0.1% (84, 61, 43, and 51, denoted with asterisks), where both ampliCan and CRISPResso2 determined the sites as ≤0.1%. Error bars represent confidence intervals of CRISPECTOR results. This activity was validated by human examination of the individual actual reads (see “Discussion” and Supplementary Note 2). c Tool comparison for the active off-target site classification. Off-target site classification results for CRISPECTOR, CRISPResso2 (CS2), and ampliCan (Amp). In the left column squares, the number of instances with agreement and disagreement in the classification task are depicted. Each square refers to a comparison of two tools. P (Positive) and N (Negative) stand for active and inactive sites, respectively. The two pie charts depict CRISPECTOR and CRISPResso2 classification results on the 19 validated sites. TP, TN, FP, and FN denote true-positive, true-negative, false-positive, and false-negative, respectively. True state determined by the actual reads.