Gene editing by SSB/CRISPR-Cas9 ribonucleoprotein in bacteria.

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• CRISPR-Cas9 ribonucleoprotein (RNP) gene editing is established in plants and animals.
• Its application for bacterial gene editing remains largely unexplored.

Purpose of the Study:

• To develop and evaluate a novel SSB/CRISPR-Cas9 RNP gene editing system for bacteria.
• To demonstrate efficient non-homologous and homologous recombination gene editing in bacterial species.

Main Methods:

• Constructed an SSB/CRISPR-Cas9 RNP system for editing the upp gene in *Escherichia coli*, *Pseudomonas*, and *Bacillus subtilis*.
• Introduced RNP and SSB into bacterial protoplasts for non-homologous recombination.
• Introduced RNP, SSB, and oligodeoxynucleotide (ODN) donors (single-stranded or double-stranded) for homologous recombination.

Main Results:

• Achieved non-homologous recombination efficiencies of 9.75%, 5.02%, and 8.37% in *E. coli*, *Pseudomonas*, and *B. subtilis*, respectively.
• Demonstrated homologous recombination knockout efficiencies of 45.11%, 30.13%, and 27.18% using ssODN donors.
• Achieved homologous recombination replacement efficiencies of 35.94%, 22.46%, and 19.08% using dsODN donors carrying a tetracycline resistance gene.

Conclusions:

• The SSB/CRISPR-Cas9 RNP system provides an efficient, simple, and rapid method for bacterial genome editing.
• This system eliminates the need for plasmids in bacterial gene editing.
• This research represents the first report of RNP-based gene editing in bacteria.