Target DNA mutagenesis-based fluorescence assessment of off-target activity of the CRISPR-Cas9 system

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• The CRISPR/Cas9 system enables precise DNA cleavage but can cause off-target mutations.
• Understanding sgRNA-DNA interactions at off-target sites is crucial for safety.
• A direct cell-based assay for detecting Cas9 activity at off-target sites was lacking.

Purpose of the Study:

• To develop a rapid, cell-based fluorescence assay for detecting Cas9 activity at off-target DNA sites.
• To investigate the impact of nucleotide mismatches on Cas9 binding and activity.
• To evaluate the specificity of Cas9 nucleases and high-fidelity variants.

Main Methods:

• Development of a target DNA mutagenesis-based fluorescence assay.
• Direct detection of Cas9 nuclease activity in living cells.
• Assessment of Cas9 tolerance to nucleotide mismatches near PAM sites.

Main Results:

• Cas9 nuclease exhibits low tolerance to nucleotide mismatches in the binding region adjacent to PAM sites.
• A trade-off between Cas9 activity and specificity was observed.
• Comparison with a high-fidelity Cas9 variant highlighted specificity differences.

Conclusions:

• The developed fluorescence assay enables direct detection of Cas9 off-target activity in cells.
• Understanding mismatch tolerance is key to minimizing off-target mutations.
• Combining computational predictions with this assay can guide safer genome engineering strategies.