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CRISPR-Cas12a-Assisted Recombineering in Bacteria
Mei-Yi Yan1, Hai-Qin Yan2, Gai-Xian Ren1
1MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, and Center for Tuberculosis Research, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Applied and Environmental Microbiology
|June 25, 2017
Summary
This study introduces a CRISPR-Cas12a-assisted recombineering system for efficient bacterial genome editing. The method enables markerless and scarless genetic modifications, including mutations and replacements, in various bacterial species.
Area of Science:
- Microbiology
- Molecular Biology
- Genome Engineering
Background:
- CRISPR-Cas systems, including CRISPR-Cas9, are established tools for bacterial genome editing.
- CRISPR-Cas12a (Cpf1) offers an alternative CRISPR-Cas system with distinct target recognition properties.
- Recombineering is a powerful technique for genetic manipulation in bacteria.
Purpose of the Study:
- To develop and optimize a CRISPR-Cas12a-assisted recombineering system for bacterial genetic manipulation.
- To demonstrate the system's utility for generating various genetic modifications in key bacterial species.
- To establish an efficient method for markerless and scarless genome editing in bacteria.
Main Methods:
- Development and optimization of a CRISPR-Cas12a-assisted recombineering workflow.
- Application of the system for generating point mutations, deletions, insertions, and gene replacements.
- Testing the system in *Escherichia coli*, *Yersinia pestis*, and *Mycobacterium smegmatis*.
Main Results:
- The CRISPR-Cas12a-assisted recombineering system successfully facilitated diverse genetic modifications.
- Markerless and scarless mutations were efficiently generated on bacterial chromosomes and native plasmids.
- The system proved effective across multiple bacterial species, including pathogens.
Conclusions:
- CRISPR-Cas12a-assisted recombineering provides an efficient and versatile platform for bacterial genome editing.
- This approach overcomes limitations of antibiotic resistance gene insertion, enabling scarless modifications.
- The findings offer guidance for implementing Cas12a-mediated genome editing in bacteria.
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