A strategy for Cas13 miniaturization based on the structure and AlphaFold

Feiyu Zhao, Tao Zhang, Xiaodi Sun, Xiyun Zhang, Letong Chen, Hejun Wang, Jinze Li, Peng Fan, Liangxue Lai (), Tingting Sui () and Zhanjun Li ()
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Feiyu Zhao: Jilin University
Tao Zhang: Jilin University
Xiaodi Sun: Jilin University
Xiyun Zhang: Jilin University
Letong Chen: Jilin University
Hejun Wang: Jilin University
Jinze Li: Jilin University
Peng Fan: Jilin University
Liangxue Lai: Jilin University
Tingting Sui: Jilin University
Zhanjun Li: Jilin University

Nature Communications, 2023, vol. 14, issue 1, 1-13

Abstract: Abstract The small size of the Cas nuclease fused with various effector domains enables a broad range of function. Although there are several ways of reducing the size of the Cas nuclease complex, no efficient or generalizable method has been demonstrated to achieve protein miniaturization. In this study, we establish an Interaction, Dynamics and Conservation (IDC) strategy for protein miniaturization and generate five compact variants of Cas13 with full RNA binding and cleavage activity comparable the wild-type enzymes based on a combination of IDC strategy and AlphaFold2. In addition, we construct an RNA base editor, mini-Vx, and a single AAV (adeno-associated virus) carrying a mini-RfxCas13d and crRNA expression cassette, which individually shows efficient conversion rate and RNA-knockdown activity. In summary, these findings highlight a feasible strategy for generating downsized CRISPR/Cas13 systems based on structure predicted by AlphaFold2, enabling targeted degradation of RNAs and RNA editing for basic research and therapeutic applications.

Date: 2023
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DOI: 10.1038/s41467-023-41320-8

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