Supplementary Figure 7: Genome-wide specificity assessment of AsCas12a and AsCas12a variants.
(a) Schematic of the GUIDE-seq method31. (b, c) Comparison of the on-target mutagenesis (panel b) and GUIDE-seq dsODN tag integration (panel c) activities of AsCas12a nucleases for GUIDE-seq samples. Percent modification and tag integration assessed by T7E1 and RFLP assays, respectively; mean, s.e.m., and individual data points shown for n = 3. (d) Ratio of GUIDE-seq dsODN tag integration to overall mutagenesis for AsCas12a nucleases; data from panels b and c, mean, s.e.m., and individual data points shown for n = 3. (e, f) GUIDE-seq genome-wide specificity profiles for AsCas12a, enAsCas12a, and enAsCas12a-HF1 each paired with crRNAs targeting sites with TTTV PAMs (panel e) or non-canonical PAMs (panel f). Mismatched positions in off-target sites are highlighted in color; GUIDE-seq read counts are shown to the right of the sequences; yellow diamonds indicate off-target sites that are only supported by asymmetric GUIDE-seq reads; green circles indicate off-target sites previously identified10 for LbCas12a; alternate nucleotides in non-canonical PAMs with mean PAMDA ks > 0.005 for enAsCas12a are not colored/highlighted as mismatches; enAsCas12a-HF1 not assessed on CTTA-1, CTTC-2, or TATC-1. AsCas12a variants encode the following substitutions: enAsCas12a, E174R/S542R/K548R; enAsCas12a-HF1, E174R/N282A/S542R/K548R. References:10. Kleinstiver, B. P. et al. Genome-wide specificities of CRISPR–Cas Cpf1 nucleases in human cells. Nat. Biotechnol. 34, 869–874 (2016). 31. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).
