Nature Biotechnology: a high-fidelity Cas13 system with higher specificity and better safety

A new study has revealed that through mutation, the development of high-fidelity Cas13 variants significantly reduces collateral effect.

This work was done by researchers in D.

The CRISPR-Cas13 system is a highly efficient programmable RNA targeting tool that has been used for nucleic acid detection and RNA manipulation in various cell types and organism.

Due to this so-called collateral effect, Cas13 may randomly degrade target and non-target RNAs, making experimental design and interpretation of results difficult when using Cas13.

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

In this study, the researchers designed a dual fluorescent reporter plasmid system (EGFP and mCherry) to examine the collateral effects of Cas13 in mammalian cell.

The researchers then attempted to engineer Cas13d (RfxCas13d, a Cas13d from Ruminococcus flavus XPD3002) by mutagenesis, based on a novel well-designed dual-fluorescent reporter system, including one containing EGFP, mCherry, and EGFP targeting Single plasmids of gRNA, and each Cas13 variant, screened for the variant with minimal incidental impac.

Hui Yang's team designed and generated a mutant library of Cas13d variants and transfected them into HEK293 cells individuall.

Due to its high specificity for RNA degradation, the variant N2V8, termed high-fidelity Cas13d (hfCas13d), was used for further characterization, including whole-transcriptome off-target analysis using RNA sequencin.

Hui Yang's team discovered the CRISPR-Cas13X system in 2021, which is currently the smallest (only 775 amino acids) RNA editing too.

article title

High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects