Fig. 1

Generation of AsCas12a variants with increased specificity by weakening non-specific DNA contacts. a Schematic of wild-type AsCas12a interaction with the target DNA-sgRNA duplex. b The mutation sites of the AsCas12a variants. c Tag-seq-based comparative analyses of wild-type AsCas12a (WT), AsCas12a variant KA (KA), and AsCas12a variant KK (KK) with seventeen sgRNAs targeting nine genes (also see Additional file 2: Figure S1e). The sgRNA was shown on the top and the on-target and the off-target cleavages were displayed without or with mismatches to the sgRNA reference by color highlighting. Sequencing read counts were shown to the right of each site. d Total number of off-target sites detected with the seventeen sgRNAs. e Specificity Index (value was calculated by the ratio of total on-target reads to the on-target reads plus the off-target reads within the seventeen sites). f Normalization of on-target activity of KK and KA to wild-type AsCas12a