Fig. 6: mbPE can introduce edits at sites distal from the PAM.

A Schematic overview of the RTT design strategy used to edit bases distal from the PAM site. Every pegRNA introduces a single PAM mutation (G to C) and a second, purine to pyrimidine (or vice versa) transversion introduced at varying distances from the PAM sequence. B Fraction of correctly edited luc alleles and pegRNA read counts over the total reads in that sample of DNA extracted from cells grown in the absence (control) or presence of aTc. The average fraction of pegRNA reads for the substitutions is shown as dots on the right Y-axis. Edits are observed up to 91 bp from the PAM site. Resulting means of four biological replicates are plotted. C Validation of the maximum editing distance through individually cloned pegRNAs. The mutations conferred by the pegRNAs all encode the G to C PAM mutation and a second mutation at varying distances. The 33, and 45 bps substitutions from the PAM site, confer a premature stop codon, whilst the pegRNAs encoding mutation either 72 or 91 bps from the PAM site resulted in a glycine to cysteine (72 bps) or isoleucine to asparagine (91 bps) substitution (n = 21 each). The percentages indicate the fraction of clones that were phenotypically observed to be edited. Clones with an RLU/OD value below 3 × 10³ were considered to be edited. The mutation at 91 nts (*) could only be confirmed by sequencing individual clones, as the amino acid change does not affect luciferase function. This showed an editing efficiency of 4.8 % (1 out of 21 tested colonies) with the correct genome edit.