Supplementary Figure 9: On-target activity assessment of AsCas12a, enAsCas12a, and enAsCas12a-HF1. | Nature Biotechnology

Supplementary Figure 9: On-target activity assessment of AsCas12a, enAsCas12a, and enAsCas12a-HF1.

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

Supplementary Figure 9

(a) Time-course in vitro cleavage reactions of Cas12a orthologs and variants conducted at 37, 32, and 25 °C (left, middle, and right panels, respectively) using the PAMDA site 1 substrate. Curves were fit using a one phase exponential decay equation; mean and error bars represent s.e.m for n = 3. (b, c) Assessment of the on-target activities of AsCas12a, enAsCas12a, and enAsCas12a-HF1 by plasmid electroporation into U2OS cells on target sites harboring TTTV PAMs (panel b) or non-canonical VTTV, TATV, TTCV, and TTTT PAMs (panel c). Percent modification assessed by T7E1 assay; mean, s.e.m., and individual data points shown for n = 3. (d, e, f) Assessment of the on-target activities of AsCas12a, enAsCas12a, and enAsCas12a-HF1 when delivered as RNPs into HEK2923 cells (panels d and e) and primary T cells (panel f). The mean activities for each site in panel d are shown in panel e. Percent modification assessed by T7E1 assay; mean, s.e.m., and individual data points shown for n = 3; ns, P > 0.05; *, P < 0.05 (Wilcoxon signed-rank, two-tailed; P values in Supplementary Table 8). AsCas12a variants encode the following substitutions: enAsCas12a, E174R/S542R/K548R; enAsCas12a-HF1, E174R/N282A/S542R/K548R.

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