An engineered xCas12i with high activity, high specificity and broad PAM range

Zhang, H.; Kong, X.; Xue, M.; Wang, Z.; Wei, Y.; Wang, H.; Zhou, J.; Zhang, W.; Xu, M.; Shen, X.; Li, J.; Hu, J.; Zhong, N.; Zhou, Y.; Yang, H.

doi:10.1101/2022.06.15.496255

bioRxiv · 10.1101/2022.06.15.496255

An engineered xCas12i with high activity, high specificity and broad PAM range

Zhang, H.·Kong, X.·Xue, M.·Wang, Z.·Wei, Y.·Wang, H.·Zhou, J.·Zhang, W.·Xu, M.·Shen, X.·Li, J.·Hu, J.·Zhong, N.·Zhou, Y.·Yang, H.

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Abstract

The type-V CRISPR effector Cas12i, with its smaller size, short crRNA guiding, and self-processing features, is a potentially versatile genome editing tool. By screening Cas12i proteins from a metagenomic database, we identified a natural variant with high activity in mammalian cells, named as xCas12i. We further engineered the PAM-interacting, REC, and RuvC domains for enhanced cleavage activity and specificity. This variant, named as high-fidelity Cas12Max, exhibited robust genome editing activity and minimal off-target activity with a broad 5-TN recognition profile. With the fusion of deaminase TadA8e and further optimization of xCas12i, the base editor dCas12i-Tad8e also showed the high editing efficiency. This study provides highly efficient and specific tools for gene therapy.

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Zhang, H., Kong, X., Xue, M., Wang, Z., Wei, Y., Wang, H., Zhou, J., Zhang, W., Xu, M., Shen, X., Li, J., Hu, J., Zhong, N., Zhou, Y., Yang, H.. 2022-06-15. An engineered xCas12i with high activity, high specificity and broad PAM range. https://doi.org/10.1101/2022.06.15.496255

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