Poster Presentation 51st Lorne Proteins Conference 2026

Substrate bound cryoEM structures reveal CRISPR-Cas13 specifically cleaves tRNA to provide bacterial immunity. (#209)

Brooke K Hayes 1 , Roland W Calvert 1 , Nathan Chai 1 , Yongyi Peng 2 , Enzo Huang 1 3 , Hari Venugopal 4 , Cyntia Taveneau 1 , Jovita D Silva 1 , Sefi Rosenbluh 1 , Chen Davidovich 1 , Chris Greening 2 , Rebecca S Bamert 1 , Gavin J Knott 1
  1. Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, Australia
  2. Microbiology, Monash University, Clayton, VIC, Australia
  3. Monash Proteomics and Metabolomics Platform, Monash University, Melbourne, VIC, Australia
  4. Ramaciotti Centre for Cryo-Electron Microscopy, Monash University, Melbourne, VIC, Australia

Bacteria are in constant competition with highly infectious bacteriophages. To protect themselves from phage infection, bacteria can use an RNA guided adaptive immune system, CRISPR-Cas13. During phage infection, activated CRISPR-Cas13 degrades both foreign and host RNA inducing stalling or death of the infected cell crucially preventing phage propagation. Given that CRISPR-Cas13 is activated by specific viral RNA transcripts, it has numerous applications for diagnosing human viral infections. However, to further develop these diagnostics there must be rigorous understanding of CRISPR-Cas13 biology. Many studies have dissected how CRISPR-Cas13 is activated, yet little is known about binding and cleavage of substrate RNA, and how stalling-based immunity is mediated.

 

Using biochemical techniques and RNA sequencing, we show that Cas13 from three different bacterial species specifically cut tRNA which likely leads to translational stalling and bacterial cell dormancy. This contrasts the common belief that Cas13 RNA cleavage is non-specific. Using these data, we determined five high-resolution cryoEM structures of a biotechnologically relevant Cas13 (LbuCas13a) during different stages of substrate tRNA capture and cleavage. These structures highlighted key residues that mediate substrate recognition and binding. To confirm the importance of these residues, we designed and tested the activity of a suite of LbuCas13a mutants. We used these data to successfully engineer Cas13 variants with higher catalytic efficiency and altered RNA specificity, both applicable for diagnostic application.

 

Together, this study shows for the first time the mechanism of CRISPR-Cas13 mediated bacterial cell dormancy and immune response. We provide foundational data for determining the structural features that drive substrate specificity of the Cas13 ribonuclease and use this knowledge to engineer more efficient Cas13 variants. This broadens our understanding of how CRISPR-Cas13 mediates bacterial immunity and allows for the development of innovative tools for rapid and sensitive CRISPR-Cas13 based diagnostics.