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<p>Cas13 is a class II type VI CRISPR effector that relies on guide RNA (gRNA) complementarity to target an RNA transcript for cleavage. Despite its ability to target any desired RNA sequence, the Cas13 endoribonuclease frequently exhibits nonspecific collateral RNase activity in bacteria and human cells. Since the collateral activity of Cas13 has not been demonstrated in plant systems, we here developed a fluorescence-based experimental pipeline using <i>Arabidopsis thaliana</i> protoplasts to analyze the target RNA cleavage and collateral RNA degradation activities of RfxCas13d, the most widely used Cas13 protein. We found that when used together with a gRNA and target RNA, RfxCas13d not only cleaved the target RNA but also induced the collateral degradation of non-target RNAs. The collateral activity of Cas13 was particularly evident in the case of mRNAs produced from a recombinant plasmid co-transfected with the <i>Cas13-gRNA</i> construct, while endogenous mRNAs remained unaffected. Thus, our findings suggest that Cas13 exhibits collateral RNase activity in plants, and this characteristic of Cas13 may limit its application for plant genome editing.</p>

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RfxCas13d Exhibits Collateral RNA Degradation Activity in Plant Systems

  • 期刊论文

  • 发表日期：

    2025年5月21日

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  • Jeong-Won Nam,
  • Yeong Yeop Jeong,
  • Pil Joon Seo

摘要

Cas13 is a class II type VI CRISPR effector that relies on guide RNA (gRNA) complementarity to target an RNA transcript for cleavage. Despite its ability to target any desired RNA sequence, the Cas13 endoribonuclease frequently exhibits nonspecific collateral RNase activity in bacteria and human cells. Since the collateral activity of Cas13 has not been demonstrated in plant systems, we here developed a fluorescence-based experimental pipeline using Arabidopsis thaliana protoplasts to analyze the target RNA cleavage and collateral RNA degradation activities of RfxCas13d, the most widely used Cas13 protein. We found that when used together with a gRNA and target RNA, RfxCas13d not only cleaved the target RNA but also induced the collateral degradation of non-target RNAs. The collateral activity of Cas13 was particularly evident in the case of mRNAs produced from a recombinant plasmid co-transfected with the Cas13-gRNA construct, while endogenous mRNAs remained unaffected. Thus, our findings suggest that Cas13 exhibits collateral RNase activity in plants, and this characteristic of Cas13 may limit its application for plant genome editing.