Fig. 2: Tailoring a cytidine base editor for multiplex genome modifications in P. putida.
A Upon cytidine deamination, the paired guanidine is converted to adenine and thymidine, or uridine is recognized by uracil-DNA glycosylase (UNG), an activity blocked by the UNG inhibitor (UGI). B Incorporation of ugi to editing plasmids (pBEC) towards increasing editing efficiency. In a subsequent step, cas6f was introduced for processing multiple gRNAs (pMBEC). Note that x is a placeholder identifying antibiotic resistances (AbR). The vectors are carrying furthermore a vegetative origin of replication (oriV) and the sucrose counter selectable marker sacB. C Processing of the RNA cassette by Cas6. Each gRNA harbors a 3’-recognition site for Cas6; Cas6 remains attached to gRNAs upon cleavage. D Protospacers used to investigate positional and preceding base effects. All positions within the editing window are tested for editing with any of the four bases proceeding. PAM, protospacer adjacent motif. E Editing efficiency at any protospacer position depending on preceding base and F editing efficiency depending on position in the protospacer and preceding base, quantified with and without ugi. Mean values of two independent biological experiments are presented; dots represent data per experiment with at least eight colonies analyzed per replicate. Source data underlying panels E, F are provided as a Source Data file.
