Make-or-break prime editing for genome engineering in Streptococcus pneumoniae

Monica Rengifo-Gonzalez, Maria-Vittoria Mazzuoli, Axel B. Janssen, Anne-Stéphanie Rueff, Jessica Burnier, Xue Liu () and Jan-Willem Veening ()
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Monica Rengifo-Gonzalez: CH-
Maria-Vittoria Mazzuoli: CH-
Axel B. Janssen: CH-
Anne-Stéphanie Rueff: CH-
Jessica Burnier: CH-
Xue Liu: Shenzhen
Jan-Willem Veening: CH-

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: Abstract CRISPR-Cas9 has revolutionized genome engineering by allowing precise introductions of DNA double-strand breaks (DSBs). However, genome engineering in bacteria is still a complex, multi-step process requiring a donor DNA template for repair of DSBs. Prime editing circumvents this need as the repair template is indirectly provided within the prime editing guide RNA (pegRNA). Here, we developed make-or-break Prime Editing (mbPE) that allows for precise and effective genetic engineering in the opportunistic human pathogen Streptococcus pneumoniae. In contrast to traditional prime editing in which a nicking Cas9 is employed, mbPE harnesses wild type Cas9 in combination with a pegRNA that destroys the seed region or protospacer adjacent motif. Since most bacteria poorly perform template-independent end joining, correctly genome-edited clones are selectively enriched during mbPE. We show that mbPE is RecA-independent and can be used to introduce point mutations, deletions and targeted insertions, including protein tags such as a split luciferase, at selection efficiencies of over 93%. mbPE enables sequential genome editing, is scalable, and can be used to generate pools of mutants in a high-throughput manner. The mbPE system and pegRNA design guidelines described here will ameliorate future bacterial genome editing endeavors.

Date: 2025
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DOI: 10.1038/s41467-025-59068-8

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