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PreprintCited by 12CRISPR and Genetic EngineeringRNA regulation and diseaseRNA Interference and Gene Delivery

Collateral cleavage of 28s rRNA by RfxCas13d causes death of mice

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Li, Yunfei, Xu, Junjie, Guo, Xuefei, Li, Zhiwei, Cao, Lili, Liu, Shengde, Guo, Ying, Wang, Guodong +16 more·2022·bioRxiv (Cold Spring Harbor Laboratory)
DOI 10.1101/2022.01.17.476700
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Abstract

Summary: 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. Here, we found that knocking down gene expression using RfxCas13d in the adult brain neurons caused death of mice, which was not resulted from the loss of target gene function or off-target effects. Mechanistically, we showed that RfxCas13d exhibited 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 (IEG) pathway. These results provide new mechanistic insights into the collateral activity of RfxCas13d and warn that the biosafety of CRISPR-Cas13 system needs further evaluation before applying it to clinical treatments.

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  • Significant unintended editing has been reported for CasRx, hampering its clinical progress.

    “however, significant unintended editing has been reported hampering its clinical progress (8, 9)”

    From Characterization of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina (2024) · cites this paper as [8]

    The Abstract reports unintended collateral cleavage of 28S rRNA by RfxCas13d, mouse deaths, and a need for safety evaluation before clinical treatment. The Discussion likewise warns about treatment safety. The paper describes RNA cleavage, not unintended editing, and does not establish that clinical progress has actually been hampered. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • RNA editing with CRISPR-Cas13 has emerged as a powerful tool for transient control of gene expression, although recurrent reports of collateral activity are a hurdle for clinical translation.

    “RNA editing with CRISPR-Cas13 has emerged as a powerful tool for transient control of gene expression, although recurrent reports of collateral activity are a hurdle for clinical translation.”

    From Characterization of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina (2024) · cites this paper as [8]

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Authors
Li, Y., Xu, J., Guo, X., Li, Z., Cao, L., Liu, S., Guo, Y., Wang, G., Luo, Y., Zhang, Z., Wei, X., Zhao, Y., Liu, T., Wang, X., Xia, H., Kuang, M., Guo, Q., Li, J., Chen, L., Wang, Y., Li, Q., Wang, F., Liu, Q., & You, F.
Affiliations
  • Peking University (CN)
  • Beijing Normal University (CN)
  • Hebei University (CN)
  • Chinese Academy of Sciences (CN)
  • Peking University Cancer Hospital (CN)
  • National Institute of Biological Sciences, Beijing (CN)
Corresponding author
  • Liu, Qinghua
  • You, Fuping
ORCID
Venue
bioRxiv (Cold Spring Harbor Laboratory)
Publisher
openRxiv
Published
2022-01-18
DOI
10.1101/2022.01.17.476700
OpenAlex
W4205218580
Cited by
12
References
2
Citations by year
2024: 1 · 2023: 3 · 2022: 8
Type
posted content
Language
en
Open access
Locations
bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
Funders
  • National Natural Science Foundation of China (2020YFA0707800, 31570891, PKU2020LCXQ009, 32022028, 81991505, 31872736, 2016YFA0500300)
  • Peking University (PKU2020LCXQ009)
  • National Key Research and Development Program of China (32022028, 31570891, 81991505, PKU2020LCXQ009, 31872736, 2020YFA0707800, 2016YFA0500300)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
CRISPR-Cas13, collateral cleavage, collateral activity, mouse lethality, brain neurons, translation attenuation, immediate early genes, biosafety

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