biostudies-literatureRengifo-Gonzalez MSwiss National Science FoundationEuropean Research Council3796https://www.ebi.ac.uk/biostudies/studies/S-EPMC12015366biostudies-literatureUnknown16(1)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.Nature communicationsMake-or-break prime editing for genome engineering in Streptococcus pneumoniae.PMC12015366NCCR AntiResist 51NF40_180541200792771534-PneumoCaTChERTMPFP3_210202192517310030_200792310030771534Liu XMazzuoli MVBurnier JJanssen ABVeening JWRengifo-Gonzalez MRueff ASfalseMake-or-break prime editing for genome engineering in Streptococcus pneumoniae.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.2025-01-01T00:00:00Z2025 Apr2026-06-01T12:54:47.496Z2025-07-03T03:05:02.581ZS-EPMC120153664026327410.1038/s41467-025-59068-8