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Make-or-break prime editing for genome engineering in Streptococcus pneumoniae.
Monica Rengifo-Gonzalez1, Maria-Vittoria Mazzuoli1, Axel B Janssen1
1Department of Fundamental Microbiology, Faculty of Biology and Medicine, University of Lausanne, Biophore Building, CH-, Lausanne, Switzerland.
Nature Communications
|April 22, 2025
Summary
We developed make-or-break Prime Editing (mbPE), a novel CRISPR-Cas9 tool for precise bacterial genome engineering. This method simplifies genetic modifications in Streptococcus pneumoniae, enhancing future research and applications.
Area of Science:
- Microbiology
- Molecular Biology
- Genome Engineering
Background:
- CRISPR-Cas9 enables precise DNA double-strand breaks (DSBs) but bacterial genome engineering remains complex.
- Traditional methods require donor DNA templates for DSB repair, complicating bacterial genetic manipulation.
- Prime editing offers an alternative by integrating repair templates into the prime editing guide RNA (pegRNA).
Purpose of the Study:
- To develop a novel, efficient, and simplified genome engineering system for Streptococcus pneumoniae.
- To overcome the limitations of existing bacterial genome editing techniques.
- To establish a versatile platform for introducing various genetic modifications in bacteria.
Main Methods:
- Development of make-or-break Prime Editing (mbPE) utilizing wild type Cas9 and a modified pegRNA.
- The pegRNA in mbPE is designed to disrupt the seed region or protospacer adjacent motif, enhancing selection.
- Exploitation of bacteria's inefficient template-independent end joining for selective enrichment of edited clones.
Main Results:
- mbPE allows precise point mutations, deletions, and targeted insertions, including protein tags, in Streptococcus pneumoniae.
- Achieved high selection efficiencies exceeding 93% for correctly genome-edited clones.
- Demonstrated mbPE's RecA-independence, scalability, and suitability for high-throughput mutant pool generation and sequential editing.
Conclusions:
- mbPE offers a simplified, effective, and versatile method for bacterial genome engineering.
- The system streamlines genetic modifications in Streptococcus pneumoniae, facilitating future research.
- mbPE and its associated pegRNA design guidelines are expected to advance bacterial genome editing endeavors.
