Mechanisms of adaptive immunity in RNA-targeting CRISPR-Cas13 systems

Summary CRISPR-Cas systems are prokaryotic adaptive immune mechanisms that provide robust defense against foreign genetic invaders through sequence-specific targeting by Cas effectors guided by CRISPR RNAs. Programmable Cas nucleases have revolutionized research and biomedicine. Among evolutionarily and mechanistically distinct CRISPR-Cas, type VI systems stand out for employing single-subunit Cas13 effectors, which exclusively recognize and cleave RNA. These RNA-targeting Cas13 nucleases have significantly expanded the CRISPR toolkit, enabling applications in RNA sensing, knockdown, editing, and highly sensitive diagnostics. Upon target RNA binding, Cas13 RNase is activated, leading to target RNA cleavage and collateral cleavage of non-complementary RNAs. While target RNA primarily serves as an activator of Cas13 RNase, collateral RNA cleavage triggers a broad cellular response, inducing cell dormancy and effectively impeding the spread of invaders through the bacterial population. However, the extent and specificity of collateral cleavage and the cellular processes it disrupts remain poorly understood. In addition, little is known about immunization in these RNA-centric systems. We recently showed that the principal form of collateral RNA damage elicited by Cas13a from Leptotrichia shahii is the cleavage of tRNAs at anticodon loop, leading to translation inhibition and cell growth arrest. Whether tRNA cleavage is unique to this Cas13 protein or shared across other Cas13 homologs remains unknown, raising fundamental questions about defense mechanisms triggered by collateral RNA cleavage. Using microbiological, genetic, and high-throughput sequencing approaches combined with biochemical and structural analyses, we will 1) systematically investigate the collateral activity of a broad panel of Cas13 homologs representing distinct clades across the Cas13 phylogeny, including miniature ancestral Cas13 proteins, to determine their cleavage preferences and mechanistic details of RNA recognition and cleavage; 2) define the defense mechanisms induced by collateral RNA cleavage, with a focus on understanding translation inhibition triggered by Cas13-mediated tRNA cleavage; 3) explore distinct spacer acquisition strategies in RNA-targeting type VI CRISPR-Cas systems. This research will substantially advance our understanding of the biological functions of CRISPR-Cas13 systems and guide the development of Cas13-based technologies. Project Number: 1R35GM164240-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Ekaterina Semenova | Institution: RUTGERS, THE STATE UNIV OF N.J., PISCATAWAY, NJ | Award Amount: $487,500 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MGG-D (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11329880