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Updated: Aug 12, 2025
Ubiquitous and Tissue-specific RNA Targeting in Drosophila Melanogaster using CRISPR/CasRx
Published on: February 5, 2021
The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model
Yunfei Li1, Junjie Xu2,3, Xuefei Guo4
1Department of Immunology, School of Basic Medical Sciences, Beijing Key Laboratory of Tumor Systems Biology, Institute of Systems Biomedicine, Peking University Health Science Center, Beijing, 100191, China. 1711110048@bjmu.edu.cn.
Background:
The CRISPR-Cas13 system is an RNA-guided RNA-targeting system and has been widely used in transcriptome engineering with potentially important clinical applications. However, it is still controversial whether Cas13 exhibits collateral activity in mammalian cells.
Results:
Here, we find that knocking down gene expression using RfxCas13d in the adult brain neurons caused death of mice, which may result from the collateral activity of RfxCas13d rather than the loss of target gene function or off-target effects. Mechanistically, we show that RfxCas13d exhibits collateral activity in mammalian cells, which is positively correlated with the abundance of target RNA. The collateral activity of RfxCas13d could cleave 28s rRNA into two fragments, leading to translation attenuation and activation of the ZAKα-JNK/p38-immediate early gene pathway.
Conclusions:
These findings provide new mechanistic insights into the collateral activity of RfxCas13d in mammalian cells and warn that the biosafety of the CRISPR-Cas13 system needs further evaluation before application to clinical treatments.
Insights
The CRISPR-Cas13 system
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas13 is an RNA-guided RNA-targeting system used in transcriptome engineering.
- Potential clinical applications exist, but Cas13's collateral activity in mammalian cells remains debated.
Purpose of the Study:
- Investigate the collateral activity of RfxCas13d in mammalian cells.
- Determine the mechanism behind RfxCas13d-induced lethality in mice.
Main Methods:
- Gene knockdown using RfxCas13d in adult brain neurons of mice.
- Analysis of RfxCas13d collateral activity and its correlation with target RNA abundance.
- Investigation of RNA cleavage products and downstream cellular pathways.
Main Results:
- RfxCas13d-mediated gene knockdown in mice led to lethality, potentially due to collateral activity.
- RfxCas13d exhibits collateral activity in mammalian cells, increasing with target RNA levels.
- Collateral activity cleaves 28s rRNA, causing translation attenuation and activating the ZAKα-JNK/p38 pathway.
Conclusions:
- RfxCas13d possesses collateral activity in mammalian cells, impacting vital cellular processes.
- The findings highlight the need for further biosafety evaluations of CRISPR-Cas13 systems for clinical use.
