Supplementary Figure 3: Assessment of the improved targeting range of enAsCas12a in human cells. | Nature Biotechnology

Supplementary Figure 3: Assessment of the improved targeting range of enAsCas12a in human cells.

From: Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing

Supplementary Figure 3

(a, b) Comparison of the activities of E174R/S542R and E174R/S542R/K548R AsCas12a on endogenous sites in human cells bearing non-canonical VTTN and TTCN PAMs (panel a), or TATN PAMs (panel b). (c) Comparison of the activities of wild-type, E174R/S542R, and E174R/S542R/K548R AsCas12a on sites with TTTT PAMs. (d) Activity of wild-type AsCas12a on sites with TTCN or TATN PAMs. (e, f) Activity of the E174R/S542R/K548R variant against sites with TGTV PAMs (panel e) or additional sites with various non-canonical PAMs (panel f). (g) Correlation between the PAMDA rate constant and mean modification in human cells for the PAMs tested in panels a-e. The gray shaded box indicates an arbitrary PAMDA rate constant threshold of 0.005 (or 10−2.25) roughly predictive of activity in human cells. (h) Summary of targetable PAMs for enAsCas12a. Tiers of PAMs: 1, high-confidence PAM (mean k > 0.01, mean percent modified > 20%); 2, medium confidence PAM (mean k > 0.005, mean percent modified > 10%); 3, low activity or discrepant PAM (mean percent modified < 10% or discrepancy between mean k and percent modified). See also Supplementary Table 2. (i) Influence of the +1 (most PAM proximal) base identity on the activities of wild-type AsCas12a and enAsCas12a when targeting TTTN PAMs. The mean activities of the 26 TTTN PAM sites from Supplementary Figs. 3c and 4a are shown, with black bars representing the mean; ns, P > 0.05 (Mann–Whitney, two-tailed; P values in Supplementary Table 8); **, P < 0.01 (Wilcoxon signed-rank, two-tailed; P values in Supplementary Table 8). (j) Influence of the +1 base identity on the activity of enAsCas12a when targeting sites with non-canonical TTTT, VTTV, TTCN, and TRTV PAMs. The mean activities of the 92 sites from Supplementary Figs. 3a-c, and 3e are shown, with black bars representing the mean; ns, P > 0.05 (Mann–Whitney, two-tailed; P values in Supplementary Table 8). (k) Impact of percent GC content on the activity of enAsCas12a when targeting sites bearing canonical or non-canonical PAMs (TTYN, VTTV, and TRTV). The mean activities of the 113 sites from Supplementary Figs. 3a-c, 3e, and 4a are binned according to GC content and shown in this panel as box and whisker plots in gray (min, max, median, and quartiles shown), and black bars representing the mean. (l) Sequence logos of targets sites (8 nt PAM and 28 nt spacer) examined with enAsCas12a binned based on mean percent modification. Activities against sites with canonical or non-canonical PAMs (TTYN, VTTV, and TRTV) are from Supplementary Figs. 3a-c, 3e, and 4a. (m) PAMDA data to examine potential -5 PAM position preference exhibited by wild-type AsCas12a or enAsCas12a. (n) Mean modification activity for enAsCas12a grouped based on -5 PAM base identity. Activities from 87 sites with non-canonical NVTTV, NTTCN, and NTRTV PAMs (from Supplementary Figs. 3a, b, and 3e) are shown, with black bars representing the mean of that group; ns, P > 0.05 (Mann–Whitney, two-tailed; P values in Supplementary Table 8).

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