Fig. 2
Rational engineering of BhCas12b. a In vitro Cas12b reactions with differentially labeled DNA strands. A slower migrating product is observed during native PAGE separation and separation by denaturing PAGE reveals a preference for AkCas12b and BhCas12b to preferentially cut the non-target strand at lower temperatures. b Location of 10 of the 12 tested residues in the pocket between the target strand and the RuvC active site (purple). BhCas12b residues are highlighted in the structure of the highly similar BthCas12b (PDB: 5wti [10.2210/pdb5WTI/pdb]). c Indel activity of 176 BhCas12b mutations at DNMT1 (target 5) and VEGFA (target 7) normalized to wild type (gray circles). Error bars represent s.d. from n = 2 replicates. d Location of surface-exposed residues mutated to glycine. e Indel activity of 66 BhCas12b mutations at DNMT1 (target 5) and VEGFA (target 7) normalized to wild type (gray circles). Error bars represent s.d. from n = 2 replicates. f Summary of BhCas12b hyperactive variants. g Indel activity of BhCas12b variants at four target sites. Error bars represent s.d. from n = 3–6 replicates. h In vitro cleavage with increasing concentrations of BhCas12b WT and v4 variant. Gel is a representative image from n = 2 experiments. Source data are provided as a Source Data file
