Researchers design high-fidelity Cas13 variants to minimize collateral RNA targeting

A recent study published in Nature Biotechnology has revealed the development, through mutagenesis, of high-fidelity Cas13 variants that markedly reduce collateral effects. It also demonstrated the feasibility of hfCas13 for efficient on-target RNA degradation with almost no collateral damage in mammalian cells and animals.

This work was performed by researchers from Dr. YANG Hui’s lab at the Institute of Neuroscience (Center for Excellence in Brain Science and Intelligence Technology) of the Chinese Academy of Sciences, and an R&D team from HuiGene Therapeutics Co., Ltd.

The CRISPR-Cas13 system is a highly efficient tool for programmable RNA targeting and has been harnessed for nucleic acid detection and RNA manipulation in various cell types and organisms. However, concerns have been raised over the off-targeting effects of Cas13. Previous structural studies have suggested that when Cas13 binds to target RNA, the two HEPN nuclease domains may form a catalytic site on the protein surface, thus creating the potential for promiscuous cleavage of bystander RNA apart from on-target cleavage of target RNA.

Due to this so-called collateral effect, Cas13 may degrade both target and non-target RNA at random, thus making it difficult to design experiments and interpret results when using Cas13.

Therefore, engineering approaches to diminish or eliminate promiscuous RNA degradation are required for basic research and future in vivo applications of the Cas13 system.

In this study, the researchers designed a dual-fluorescent reporter (EGFP and mCherry) plasmid system to detect the collateral effects of Cas13 in mammalian cells. In this system, loss of mCherry fluorescent cells was interpreted as successful on-target editing; loss of EGFP fluorescent cells was interpreted as off-target cleavage. Results showed that either Cas13a or Cas13d may induce significant collateral effects.

The researchers then sought to engineer Cas13d (RfxCas13d, a Cas13d from Ruminococcus flavefaciens XPD3002) via mutagenesis and screen for variants with minimal collateral effects, based on a new well-designed, dual-fluorescent reporter system comprising a single plasmid containing EGFP, mCherry, and EGFP-targeting gRNA, together with each Cas13 variant.

YANG’s team designed and generated a mutagenesis library of Cas13d variants and individually transfected them into HEK293 cells. By analyzing reporter fluorescence using flow cytometry, five variants (N1V7, N2V7, N2V8, N3V7, and N15V4) exhibited a relatively low percentage of EGFP+ cells but a high percentage of mCherry+ cells, indicating high on-target activity with reduced collateral activity.

The variant N2V8, termed a high-fidelity Cas13d (hfCas13d) due to its high specificity for RNA degradation, was used for further characterization including transcriptome-wide off-target analysis using RNA sequencing. The hfCas13d showed marked reduction in the number of off-target genes when targeting several endogenous transcripts, such as PPIA. Moreover, hfCas13 variants showed no detectable collateral damage to cell lines, transgenic animals, and somatic cells, thus supporting their in vivo application.

YANG and his team identified the CRISPR-Cas13X system in 2021, the smallest (only 775 amino acids) RNA editing tool at present. In their new study, the researchers further engineered Cas13X and obtained the hfCas13X variant, which exhibits high specificity of on-target RNA degradation but minimal collateral effects. hfCas13X shows great application potential in the field of gene therapy based on RNA editing.

This work, entitled “High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects,” was published online in Nature Biotechnology on Aug. 11. TONG Huawei, HUANG Jia, XIAO Qingquan, HE Bingbing, DONG Xue and LIU Yuanhua are co-first authors with equal contribution.