Adapting prime editing with split prime editors in Escherichia coli and its application to Staphylococcus...

Prime editing is a precise and rapid genome-editing technique that modifies short DNA sequences using tailored guide RNAs. To implement this technique in bacteria, we used Prime Editor 2 (PE2) with the DeepPrime gRNA design tool and assessed its gene-editing efficiency in Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) cells. Our findings indicate that a split PE2, comprising a reverse transcriptase and two Cas9 nickase domains, exhibited gene-editing efficiency comparable to that of the intact PE2. The efficiency observed in E. coli was significantly affected by the target sites, edit type, and the presence of exonucleases. In MRSA, which serves as a model to evaluate the applicability in non-model bacterial species, Streptococcus pyogenes PE2 (SpPE2) exhibited superior performance relative to Staphylococcus aureus PE2 (SaPE2). Furthermore, the split SpPE2 lacking the reverse transcriptase successfully induced the intended mutation in MRSA. This study demonstrates the feasibility of prime editing within bacterial systems.