Background
The widespread use of CRISPR/Cas9 as a programmable genome editing tool has encountered hurdles, due to concerns about potential off‑target DNA cleavage by Cas9. While the analysis of off-target editing events has led to the development of Cas9 variants with greater sensitivity to mismatches, the underlying molecular mechanisms by which Cas9 detects—or fails to detect—mismatches remain poorly understood. Researchers at The University of Texas at Austin have utilized kinetic analysis to guide structure determination of Cas9 at different stages of mismatch surveillance and aberrant activation, using cryoelectron microscopy.
Technology overview
Through kinetic analysis and cryoelectron microscopy, researchers have uncovered a distinct, previously undescribed linear conformation of the duplex formed between the guide RNA (gRNA) and DNA target strand (TS) in the presence of PAM-distal mismatches, preventing Cas9 activation. The canonical kinked gRNA:TS duplex is a prerequisite for Cas9 activation, acting as a structural scaffold to facilitate Cas9 conformational rearrangements necessary for DNA cleavage. This research provides molecular insights into the structural mechanisms governing off-target effects of Cas9 and offers a blueprint for designing next-generation high-fidelity Cas9 variants that reduce off-target DNA cleavage while retaining efficient cleavage of on-target DNA.
Benefits
- Addresses the urgent need to minimize off-target DNA cleavage for therapeutic applications
- High-fidelity Cas9 variants offer improved mismatch discrimination.
- Enhanced specificity without severely reducing on-target DNA cleavage rates
Application
The development of next-generation high-fidelity Cas9 variants holds promise for enhancing the safety and efficacy of CRISPR-based genome editing. These variants are particularly relevant for therapeutic applications where precision and minimal off-target effects are critical. By harnessing the molecular insights gained from structural studies, researchers can refine Cas9 variants to achieve higher levels of specificity and efficiency, paving the way for safer and more effective genome editing interventions.
Opportunity
The University of Texas at Austin is seeking a Sponsored Research Agreement with the potential to commercialize SuperFi Cas9 variants for genome editing.
