Spacer Extension Reconciles Specificity and Activity in High-Fidelity Cas9 Genome Editing

Lu, Z.; Wei, R.; Zheng, R.; Shin, Y.-C.; Zhang, Q.; Li, J.; Wang, X.; Wei, Y.; Liu, B.; Chen, Y.; Zhang, H.; Chen, H.; Ma, L.

doi:10.1101/2025.01.16.633458

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bioRxiv · 10.1101/2025.01.16.633458

Spacer Extension Reconciles Specificity and Activity in High-Fidelity Cas9 Genome Editing

Lu, Z.·Wei, R.·Zheng, R.·Shin, Y.-C.·Zhang, Q.·Li, J.·Wang, X.·Wei, Y.·Liu, B.·Chen, Y.·Zhang, H.·Chen, H.·Ma, L.

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Abstract

AbstractEngineering high-fidelity CRISPR enzymes often leads to reduced cleavage activity, creating a significant hurdle in balancing nuclease specificity and efficiency for clinical applications. Here, we demonstrate that extending the spacer to 21 or 22 nucleotides restores the impaired cleavage activity of SuperFi-Cas9, an engineered high-fidelity Cas9 variant with seven mutations at the PAM-distal region. Structural and mutational analyses reveal that the spacer extension strengthens additional interactions at the PAM-distal end, stabilizing the nuclease- sgRNA-DNA complex, which appears to be disturbed due to the seven mutations. This approach not only provides a high-fidelity Cas9 with uncompromised efficiency but also introduces a novel strategy to enhance CRISPR complex stability. Our findings offer a promising avenue for precise and efficient genome editing, crucial for advancing CRISPR technologies toward clinical translation.

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Lu, Z., Wei, R., Zheng, R., Shin, Y.-C., Zhang, Q., Li, J., Wang, X., Wei, Y., Liu, B., Chen, Y., Zhang, H., Chen, H., Ma, L.. 2025-01-18. Spacer Extension Reconciles Specificity and Activity in High-Fidelity Cas9 Genome Editing. https://doi.org/10.1101/2025.01.16.633458

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