CRISPR-DNA Polymerase Assisted Targeted Mutagenesis for Regulable Laboratory Evolution

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genetics

Background:

• Targeted hypermutation tools are crucial for protein engineering and evolutionary studies.
• Existing tools have limitations in mutation window size and types of mutations possible.

Purpose of the Study:

• To develop an advanced in vivo mutagenesis system with expanded capabilities.
• To overcome limitations of current targeted hypermutation technologies.

Main Methods:

• Integration of mutagenic bacteriophage T5 or T7 DNA polymerases (DNAPs) with CRISPR-Cas9.
• Utilizing MS2-mediated recruitment for co-localization and off-target reduction.
• Employing a dTnpB-based transcriptional repression system for regulated mutagenesis.

Main Results:

• Achieved all possible nucleotide substitutions within a 2-kilobase window.
• Demonstrated a maximum mutation rate 1.1 × 10⁶-fold higher than wild-type E. coli.
• Reduced off-target mutation rates by up to 96.8% while maintaining on-target efficiency.
• Successfully applied CTRLE in E. coli, Bacillus subtilis, and Kluyveromyces lactis, showing significant mutation rate increases.
• Engineered triple-antibiotic resistance in 8 days and enhanced a specific pathway by threefold in 6 days.

Conclusions:

• CTRLE offers an efficient and versatile platform for accelerating continuous evolution in diverse microbial systems.
• The system expands the scope of targeted mutagenesis for protein and pathway engineering.
• CTRLE facilitates rapid laboratory evolution for various biotechnological applications.