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Improving the efficiency of high-fidelity Cas9 by enhancing PAM-distal interactions
Rong Zheng1,2, Zhike Lu1,2,3, Rongwei Wei3
1Westlake Laboratory, Hangzhou, China.
Abstract:
Engineering CRISPR enzymes for high fidelity often impairs cleavage activity. Meanwhile, a mechanistic understanding of why high-fidelity mutations reduce Cas9's cleavage activity remains unclear, presenting a challenge in balancing nuclease specificity and efficiency for clinical applications. In this study, we show that extending the spacer region to 21 or 22 nucleotides restores the impaired cleavage activity of SuperFi-Cas9, a high-fidelity Cas9 variant with 7 mutations in the RuvC domain at the protospacer adjacent motif (PAM)-distal region. Cryo-electron microscopy structures and mutational analyses reveal that the negatively charged mutations in a protruding loop of the RuvC domain create repulsive forces that destabilize the nuclease-single guide (sg)RNA-DNA complex. Spacer extension enhances interactions in the PAM-distal region, effectively restoring cleavage activity and balancing editing efficiency with specificity. In addition, we develop a deep learning model, AIdit-SuperFi, to predict optimal sgRNA length for high-fidelity genome editing. Our findings introduce a straightforward strategy to enhance CRISPR complex stability and provide mechanistic insights into the impaired cleavage activity of engineered high-fidelity Cas9, presenting a pathway toward precise and efficient genome editing and clinical translation of CRISPR technologies.
Insights
Extending the spacer region of high-fidelity CRISPR enzymes like SuperFi-Cas9 restores cleavage activity. This breakthrough enhances genome editing precision and efficiency for clinical applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Engineering CRISPR-Cas9 enzymes for higher fidelity often reduces their DNA cleavage efficiency.
- The precise mechanisms underlying this activity loss in high-fidelity variants are not fully understood.
- Balancing specificity and efficiency is crucial for clinical applications of CRISPR technology.
Purpose of the Study:
- To investigate why high-fidelity mutations in Cas9 impair cleavage activity.
- To identify strategies for restoring cleavage activity in high-fidelity Cas9 variants.
- To develop predictive models for optimizing CRISPR-Cas9 genome editing.
Main Methods:
- Utilized SuperFi-Cas9, a high-fidelity Cas9 variant with specific RuvC domain mutations.
- Performed cryo-electron microscopy and mutational analyses.
- Developed a deep learning model (AIdit-SuperFi) to predict optimal sgRNA length.
Main Results:
- Extending the spacer region to 21 or 22 nucleotides restored the cleavage activity of SuperFi-Cas9.
- Structural analysis revealed destabilizing repulsive forces in the RuvC domain of high-fidelity Cas9.
- Spacer extension was shown to enhance interactions in the PAM-distal region, stabilizing the complex.
- The AIdit-SuperFi model demonstrated predictive capability for sgRNA length optimization.
Conclusions:
- Spacer extension is a viable strategy to enhance CRISPR complex stability and restore cleavage activity in high-fidelity variants.
- Mechanistic insights into Cas9 activity loss provide a pathway for improved genome editing tools.
- Findings pave the way for more precise and efficient genome editing, facilitating clinical translation of CRISPR technologies.
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