Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

Area of Science:

• Microbiology
• Molecular Biology
• Synthetic Biology

Background:

• CRISPR/Cas9 and lambda (λ) recombinase-mediated recombineering are key for E. coli genome editing.
• Previous studies showed high mutagenesis efficiency but lacked data on robustness for large-scale applications.

Purpose of the Study:

• To define a robust, simple, and rapid protocol for single or multiple gene deletions in E. coli.
• To validate the robustness of CRISPR/Cas9 genome editing across a large number of targeted loci.

Main Methods:

• Optimized CRISPR/Cas9 protocol by testing guide RNA sequences, donor DNA length/concentration, and single/double-stranded donors.
• Validated optimized conditions by targeting 78 dispensable genes for deletion.

Main Results:

• Developed a protocol using double-stranded synthetic donor DNAs with consistently >10% mutagenesis efficiency.
• Achieved 100% robustness, meaning all targeted loci were successfully mutated.
• Demonstrated applicability for simultaneous gene deletions.

Conclusions:

• This work establishes the robustness of a CRISPR/Cas9 protocol for bacterial genome editing using a large sample size.
• The proposed protocol is suitable for high-throughput genome editing in synthetic biology.
• The findings have broad applicability for researchers in bacterial genome engineering.