Mismatch Intolerance of 5'-Truncated sgRNAs in CRISPR/Cas9 Enables Efficient Microbial Single-Base Genome Editing

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas9 is a powerful gene-editing tool.
• Off-target mutations due to DNA sequence similarity are a major limitation.
• Existing methods struggle with precise single-base editing.

Purpose of the Study:

• To investigate the potential of 5 runcated sgRNAs for precise single-base editing.
• To overcome the challenge of off-target mutations in CRISPR/Cas9 technology.
• To develop a method for negative selection of single-base edited targets.

Main Methods:

• Utilized 5 runcated sgRNAs with the CRISPR/Cas9 system.
• Tested the impact of single-base mismatches on target recognition.
• Employed an engineered Cas9 nuclease with an expanded PAM recognition site (5 NG).

Main Results:

• Single-base mismatches between target DNA and 5 runcated sgRNAs inhibited target recognition.
• The 5 runcated sgRNA/Cas9 complex effectively performed negative selection of single-base edited targets in microbial genomes.
• Demonstrated simple and efficient single-base editing near and far from the sgRNA 5 end.
• Achieved efficient single-base editing using an engineered Cas9 with an expanded PAM.

Conclusions:

• 5 runcated sgRNAs enhance CRISPR/Cas9 specificity by preventing off-target edits.
• This method enables precise single-base editing in microbial genomes.
• The approach holds promise for whole-genome single-base editing applications.