Supplementary Figure 8: Assessment and improvement of AsCas12a specificity.

(a) Activities of wild-type AsCas12a or variants bearing single substitutions when using crRNAs that perfectly match the on-target site or that encode single nucleotide mismatches. Percent modification assessed by T7E1 assay; mean, s.e.m., and individual data points shown for n = 3. (b) Activities of enAsCas12a variants bearing additional single amino acid substitutions, assessed as in panel a. (c) PAM preference profiles of enAsCas12a and enAsCas12a-HF1 assessed by PAMDA. The log10 rate constants are the mean of four replicates, two each against two distinct spacer sequences (see Supplementary Fig. 2d). (d) Comparison of the PAM preference profiles of enAsCas12a and enAsCas12a-HF1 across all 128 NNYN PAMs (Y = C or T). 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 (see Supplementary Fig. 3g); mean and error bars represent s.e.m for n = 4. (e) Scatterplot of the PAMDA determined rate constants for each NNNN PAM to compare the PAM preferences of enAsCas12a and enAsCas12a-HF1. AsCas12a variants encode the following substitutions: enAsCas12a, E174R/S542R/K548R; enAsCas12a-HF1, E174R/N282A/S542R/K548R.