Fig. 4: Enhancing editing specificity of FrCas9n-CBEs through high-fidelity mutations. | Nature Communications

Fig. 4: Enhancing editing specificity of FrCas9n-CBEs through high-fidelity mutations.

From: A universal and wide-range cytosine base editor via domain-inlaid and fidelity-optimized CRISPR-FrCas9

Fig. 4

a Illustration of seven amino acid mutations to alanine in the active pocket of FrCas9n. b Schematic of sgRNA-dependent off-target editing by FrCas9-CBE. c, d Editing outcomes of Single, double, and triple mutations of evoCDA-FrCas9n in the endogenous target sites. Specific editing C-T conversion rate (c) and off-target editing at 2-3 mismatch sites (d), with editing outcomes quantified using EditR and displayed in a heatmap (n = 4 biological replicates, data represent mean ± SD). e Schematic of orthogonal R-loop analysis for testing sgRNA-independent off-target effects of FrCas9-CBE. f C-to-T conversion frequencies of sgRNA-independent off-targets detected at six R-loop sites using dCas12a and corresponding sgRNAs. Bars represent the highest C-to-T activity at each R-loop site (n = 4 biological replicates, data represent mean ± SD). Statistical significance was analyzed using a two-tailed t-test: ns, p > 0.05. g Diagram illustrating genome-wide SNV mutations induced by CBEs in the E. coli Genome. h Bar chart showing the number of unpredictable SNVs and indels generated in the E. coli genome following base editing with different CBEs, as determined by whole-genome sequencing.

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