A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing.

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• CRISPR base editing facilitates bacterial genome engineering but is limited to single-nucleotide substitutions.
• A need exists for versatile genetic manipulation tools in prokaryotes that allow for larger edits.

Purpose of the Study:

• To adapt a CRISPR-Prime Editing system for versatile genetic manipulation in prokaryotes.
• To develop a DNA-free, single-nucleotide resolution toolkit for introducing substitutions, deletions, and insertions in E. coli.

Main Methods:

• Adaptation of a CRISPR-Prime Editing system for prokaryotic use.
• Application of the toolkit for genetic modifications in both plasmids and the E. coli chromosome.
• Testing of editing efficiencies for various types of genetic modifications, including deletions and insertions of different sizes.

Main Results:

• The developed CRISPR-Prime Editing toolkit successfully introduced substitutions, deletions, and insertions in E. coli with high fidelity.
• High efficiency (up to 40%) was achieved for 1-bp deletions under optimal conditions.
• Successful deletions of up to 97 bp and insertions of up to 33 bp were demonstrated, though efficiency decreased with larger fragment sizes.

Conclusions:

• This CRISPR-Prime Editing toolkit represents a significant advancement for E. coli genome engineering, offering versatility beyond base editing.
• The toolkit provides a foundation for developing similar advanced genetic manipulation systems for other bacterial species.