Make-or-break prime editing for genome engineering in Streptococcus pneumoniae.
Details
Serval ID
serval:BIB_5962FDB1DB67
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Make-or-break prime editing for genome engineering in Streptococcus pneumoniae.
Journal
Nature communications
ISSN
2041-1723 (Electronic)
ISSN-L
2041-1723
Publication state
Published
Issued date
23/04/2025
Peer-reviewed
Oui
Volume
16
Number
1
Pages
3796
Language
english
Notes
Publication types: Journal Article
Publication Status: epublish
Publication Status: epublish
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.
Keywords
Streptococcus pneumoniae/genetics, Gene Editing/methods, CRISPR-Cas Systems/genetics, Genome, Bacterial, RNA, Guide, CRISPR-Cas Systems/genetics, DNA Breaks, Double-Stranded, Genetic Engineering/methods, CRISPR-Associated Protein 9/metabolism, CRISPR-Associated Protein 9/genetics
Pubmed
Open Access
Yes
Create date
02/05/2025 11:38
Last modification date
03/05/2025 7:09