The Extremely Small Cas13j Protein Can Achieve Efficient RNA Editing In Vivo

Recently, a research team from Zhejiang University in China published a research paper titled "Compact RNA editors with natural miniature Cas13j nucleases" in the journal Nature Chemical Biology. Data-driven protease mining can use the diversity of species resources to mine "new proteins". This study used BT-IT fusion technology to conduct large-scale mining of microbial metagenomic data through intelligent mining algorithms, discovered extremely small Cas13j proteins and performed efficient in vivo RNA editing.

This study discovered a very small Cas13j protein family that can efficiently target and cut RNA through intelligent mining of large-scale metagenomic data of soil microorganisms. The protein size is only 424 aa, which is the smallest CRISPR-Cas13 system reported in nature so far. After engineering modification, a smaller enhanced eChiCas13j protein with a protein size of 340 aa was obtained. In addition, the research team developed a set of efficient and very small RNA single-base editor toolkits, which efficiently achieved C>U RNA editing in mice and realized the therapeutic application exploration of CRISPR's in vivo C>U RNA single-base editing. The discovery of the CRISPR-Cas13j system and the development of related RNA editing toolkits are of great significance for the development of gene therapy methods based on RNA editing and synthetic biology regulation methods.

This study mined very small Cas13 proteins based on the RxxxxH functional motif of Cas13 protein targeted RNA cutting through intelligent mining of large-scale metagenomic data of soil microorganisms. The study identified a new family of extremely small Cas13j proteins, including LepCas13j (529 aa) and ChiCas13j (424 aa). Among them, ChiCas13j from Chitinophagaceae is the smallest known natural Cas13 protein.

The study found that LepCas13j and ChiCas13j have efficient targeted RNA cleavage activity, showing high activity similar to CasRx, Cas13X.1, PspCas13b and Cas13bt1. The research team used ChiCas13j as the skeleton to develop a series of highly active eChiCas13j engineered proteins, and further reduced the size of the ChiCas13j protein to 340 aa, which is also the smallest engineered Cas13 protein reported so far.

Figure 1. Chi-RESCUE-S-mini3 pioneered efficient in vivo C-to-U RNA editing of PCSK9 in mice. (Li G, et al., 2024)

In addition, the research team developed a set of extremely small RNA base editing tools - Chi-REPAIRv2-mini and Chi-RESCUE-S-mini. These new RNA base editors showed extremely high A>G and C>U RNA single-base editing efficiency. The Chi-RESCUE-S-mini3 system was the first to achieve efficient C-to-U RNA editing of PCSK9 in mice via single AAV delivery. Compared with RESCUE-S-t1 and RESCUE-S-X.1, the Cas13j-based RNA editing tool Chi-RESCUE-S-mini mediated higher in vivo C>U RNA editing, significantly reduced the total cholesterol (Total Cholesterol, T-CHO) content of mice, and realized the cutting-edge application exploration of CRISPR's in vivo C>U RNA single-base editing.

Reference

Li G, et al. Compact RNA editors with natural miniature Cas13j nucleases. Nature Chemical Biology, 2024: 1-11.