A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9

Area of Science:

• Molecular Biology
• Biotechnology
• Bioinformatics

Background:

• CRISPR-Cas9 technology offers revolutionary gene editing capabilities.
• Therapeutic applications are hindered by unintended off-target mutations.
• Understanding SpCas9 off-target activity is crucial for safe clinical use.

Purpose of the Study:

• To investigate the impact of mismatches at the PAM-distal end on SpCas9 off-target activity.
• To elucidate the structural mechanisms underlying SpCas9 binding and cleavage.
• To improve the prediction of CRISPR-Cas9 off-target effects.

Main Methods:

• Designed and synthesized DNA targets with varying mismatches near the PAM sequence.
• Conducted in vitro biochemical assays and cell line-based experiments to assess Cas9 activity.
• Utilized Molecular Dynamics (MD) simulations to analyze RNA-DNA duplex stability and conformational changes.

Main Results:

• Demonstrated a strong correlation between mismatch location/nature and SpCas9 activity.
• Observed significantly reduced or abolished Cas9 activity at target sites with multiple mismatches (positions 15-18 upstream of PAM).
• MD simulations revealed that specific mismatches induce conformational instability in the RNA-DNA duplex, correlating with reduced activity.

Conclusions:

• Off-target activity of SpCas9 is strongly influenced by mismatches at the PAM-distal region.
• RNA-DNA duplex stability is a critical factor in determining SpCas9 target recognition and cleavage efficiency.
• Integrating duplex stability with binding energy calculations can enhance the prediction accuracy of CRISPR-Cas9 off-target effects.