Fig. 1: Creating an unbiased C-to-T CBE for E. coli MG1655 genome editing using FrCas9n.

a A schematic illustration of FrCas9n-CBE editing in MG1655. b The C-to-T editing frequencies at each cytosine position for APOBEC-SpCas9n-UGI, APOBEC-FrCas9n-UGI, and APOBEC-SPRY-UGI are quantified and summarized in the light red, blue, and pink lines, respectively. c Schematic of premature stop codon induction by the 5’-NNTA-3’ palindromic PAM sequence over a wider range. d The Venn diagram of the quantity and differences for genes termination in MG1655 by APOBEC-FrCas9n-UGI and SpCas9n-BE3. e Using four different deaminases to construct FrCas9n-CBE, selecting nine 22-nt original spacers containing each motif (TC, CC, AC, GC) at different genomic positions to comprehensively evaluate the impact of sequence context on targeted C editing efficiency. f–i Editing efficiency of four deaminases on targeted Cs in the range of C3-C11 with different motifs (TC, CC, AC, GC). (f) APOBEC; (g) evoAPOBEC; (h) hAID and (i) evoCDA, respectively (n = 4 biological replicates, data represent mean ± SD). j Quantitation and summary of C-to-T editing frequencies at each cytosine position of twelve endogenous loci in E. coli by four FrCas9-CBEs. k Comparing the off-target editing of four FrCas9-CBEs and SpCas9n-BE3 on predicted mismatched target sequences by Cas-offinder (n = 4 biological replicates, data represent mean ± SD). Statistical significance was analyzed using a two-tailed t-test: ns, p > 0.05.