{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Rengifo-Gonzalez M"],"funding":["Swiss National Science Foundation","European Research Council"],"pagination":["3796"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC12015366"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["16(1)"],"pubmed_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."],"journal":["Nature communications"],"pubmed_title":["Make-or-break prime editing for genome engineering in Streptococcus pneumoniae."],"pmcid":["PMC12015366"],"funding_grant_id":["NCCR AntiResist 51NF40_180541","200792","771534-PneumoCaTChER","TMPFP3_210202","192517","310030_200792","310030","771534"],"pubmed_authors":["Liu X","Mazzuoli MV","Burnier J","Janssen AB","Veening JW","Rengifo-Gonzalez M","Rueff AS"],"additional_accession":[]},"is_claimable":false,"name":"Make-or-break prime editing for genome engineering in Streptococcus pneumoniae.","description":"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.","dates":{"release":"2025-01-01T00:00:00Z","publication":"2025 Apr","modification":"2026-06-01T12:54:47.496Z","creation":"2025-07-03T03:05:02.581Z"},"accession":"S-EPMC12015366","cross_references":{"pubmed":["40263274"],"doi":["10.1038/s41467-025-59068-8"]}}