Fig. 3: Superior cellular genome editing by enFnCas9 variants.

a Schematic showing the architecture of all-in-one plasmids expressing nucleases as T2A-EGFP fusion with gRNA cloning sites. b Bar plot showing the indel events (%) plotted on the Y-axis as obtained from amplicon sequencing upon targeting 5’-NGG-3’ PAM with sgRNA containing 20-nt spacer (g20) targeting EMX1, HBB, c-Myc, RUNX1 loci, and its respective off-targets (OTs) by FnCas9, en1, SpCas9-HF1, and eSpCas9 in HEK293T cells. Untransfected cells were used as control. Error bars represent mean ± SEM of n = 3 independent biological replicates (except for c-Myc on-target of en1, n = 2) with individual values shown as dots. c Bar plot showing the indel events (%) plotted on the Y-axis as obtained from amplicon sequencing upon targeting 5’-AGG-3’ PAM containing HBB locus, and it’s off-target site (OT2) by en1, en15, and en31 with sgRNAs either containing 20-nt spacer (g20) or 21-nt spacer (g21) in HEK293T cells. Untransfected cells were used as control. Error bars represent mean ± SEM of n = 3 independent biological replicates with individual values shown as dots. d Bar plot showing the indel events (%) plotted on the Y-axis as obtained from amplicon sequencing upon targeting 5’-NGA-3’ PAM containing FANCF1 site 2, RUNX1 site 3 and ZNF629 site loci and its respective off-targets by en31 with sgRNAs either containing 20-nt spacer (g20) or 21-nt spacer (g21) in HEK293T cells. Untransfected cells were used as control. Error bars represent mean ± SEM of n = 3 independent biological replicates with individual values shown as dots. e Schematic showing the design of the plasmid donor template for targeting DCX1 locus in the HDR-mediated knock-in assay. f Box plot showing knock-in of a donor template at DCX locus by FnCas9, en1, en15, en31, SpCas9-HF1 and eSpCas9 in HEK293T cells. Data is represented as log2 fold change w.r.t. empty vectors, and analysed using one-way ANOVA, p-value is shown. The middle line within the box represents the median, the box edges represent the interquartile ranges and the whiskers indicate ± 1.5 × interquartile ranges of n = 3 independent biological replicates with individual values shown as dots. Source data are provided as a Source Data file.