Fig. 2: Engineering of Cas12a variants to improve mismatch tolerance.
From: CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels
a 3D structure of AsCas12a protein (PDB ID: 5B43) and locations of amino acid substitutions introduced during engineering. b Amino acid substitutions included in each AsCas12a variant are marked in blue. c Mismatch tolerance assay of six AsCas12a variants. The top panel shows the original sequence (OS) and mismatch sequences (M1 ~ M10) of each investigated site (Sites 1 ~ 4), with PAMs marked in blue and mismatches marked in red. The checkerboard diagrams in the middle panel show the relative fluorescence intensities of OS and M1 ~ M10 detected by different Cas12a variants, with 2E10 copies of DNA substrates and reaction for 30 min at 37 °C. The bottom histograms show the number of mismatch sample cases (M1 ~ M10) with an NF > 0.5 for each site. NF, normalized fluorescence. Source data are provided as a Source Data file.
