Multiplex Generation, Tracking, and Functional Screening of Substitution Mutants Using a CRISPR/Retron System

Area of Science:

• Molecular Biology
• Genetics
• Synthetic Biology

Background:

• Generating numerous genetic mutations is crucial for understanding gene function and screening for desired traits.
• Existing methods for large-scale mutation generation can be labor-intensive and lack efficiency in tracking mutations.

Purpose of the Study:

• To develop a novel CRISPR/retron system for high-throughput multiplexed generation of substitution mutations.
• To enable efficient tracking of introduced mutations through retron plasmid sequencing.
• To facilitate genome-scale screening of phenotypes in Escherichia coli.

Main Methods:

• Utilized a retron system for continuous donor DNA expression.
• Integrated a CRISPR/Cas9 cassette for targeted genomic cleavage.
• Applied the system to introduce multiplexed substitution mutations in the Escherichia coli genome.
• Tracked mutations by analyzing retron plasmid sequences.

Main Results:

• Successfully introduced a high-throughput of substitution mutations in the Escherichia coli genome.
• Demonstrated the ability to generate thousands of mutations in a single experiment.
• Showcased mutation tracking via retron plasmid sequencing, eliminating the need for laborious locus amplification.
• Validated the system's utility for screening phenotypes related to chemical responses and fitness.

Conclusions:

• The developed CRISPR/retron system offers an efficient and scalable method for generating and tracking multiplexed substitution mutations.
• This technology has the potential to significantly advance genome-scale screening capabilities.
• Facilitates high-throughput genetic analysis for diverse biological applications.