Nat Commun. 2023 Sep 8
Feiyu Zhao, Tao Zhang, Xiaodi Sun, Xiyun Zhang, Letong Chen, Hejun Wang, Jinze Li, Peng Fan, Liangxue Lai,Tingting Sui and Zhanjun Li
Products used in the paper Details Operation
AAV vector packaging AAV injection PackGene Biotech (China) created the AAV-mini-RfxCas13d-Pcsk9- crRNA2 expression cassettes and produced the viruses ( An AAV injection assay was performed as previously described44. Production of AAV8-RfxCas13d vectors was performed by PackGene Biotech (China).The AAV-mini-RfxCas13d-Pcsk9-crRNA2 titer was 2 × 1012 genome copies (GCs)/mL. Request Quote

Research Field: Liver, blood

Keywords: Molecular modelling, Genetic engineering, Molecular modelling, CRISPR-Cas9 genome editing

AAV Serotype: AAV8

Dose: 2E11/mouse

Routes of Administration: IV

Targeted organ: Liver, blood

Animal or cell line strain: mouse


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.

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