New CRISPR System Cas12a3 Targets tRNA to Combat Viruses

Scientists at Utah State University (USU), led by Ryan Jackson, PhD, have made significant advancements in the field of genetic engineering by discovering how two lesser-known CRISPR systems, Cas12a2 and Cas12a3, can target transfer RNA (tRNA) without damaging host cells. This research, published in the journal Nature, reveals a novel immune strategy that broadens the potential applications of CRISPR technology.

The research team has found that while traditional CRISPR systems like CRISPR-Cas9 typically target and cut DNA to disable invading pathogens, Cas12a2 and Cas12a3 focus on tRNA. This distinction is critical because tRNA is essential for protein synthesis; by inactivating tRNAs, these systems can impair viral replication without harming the host cell’s DNA.

Jackson, who is the R. Gaurth Hansen Associate Professor in USU’s Department of Chemistry and Biochemistry, emphasized the importance of this research. “We’re very focused on understanding the structure and function of the CRISPR systems we study,” he stated. The ultimate goal is to help researchers overcome barriers that could lead to therapeutic applications.

The findings suggest that Cas12a3 could enhance diagnostic tools for rapidly detecting infections such as COVID-19, influenza, and respiratory syncytial virus (RSV). The ability to test for multiple pathogens simultaneously with a single assay holds promise for improving public health responses.

In their paper, titled “RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity,” the researchers concluded that their discoveries could lead to more efficient diagnostic methods. They noted, “… reveal widespread tRNA inactivation as a previously unrecognized CRISPR-based immune strategy that broadens the application space of the existing CRISPR toolbox.”

The research highlights the role of tRNAs in the immune response to viral infections. By targeting these molecules, the Cas12a systems can halt viral protein production and prevent the spread of pathogens. This mechanism allows Cas12a3 to cleave tRNA in a precise manner, which could be harnessed to detect and target specific viruses effectively.

Jackson elaborated on the unique properties of Cas12a3, explaining that it modifies the shape of the protein to enable repeated cutting of nucleic acid targets. “When activated, Cas12a2 indiscriminately cleaves DNA, destroying all viral DNA, but collaterally killing the host cell as well. In contrast, Cas12a3 cleaves tRNAs, halting viral protein production while sparing the DNA of host cells,” he explained.

The research team utilized a variety of techniques, including cell-based assays and direct RNA sequencing, to demonstrate how target RNA recognition triggers Cas12a3 to cleave the tails of tRNAs. This process effectively drives growth arrest and blocks the spread of viruses.

The study revealed a previously uncharacterized clade of Cas nucleases referred to as Cas12a3. “After target RNA recognition, these nucleases preferentially cleave the conserved 3′ CCA tails of tRNAs to drive growth arrest and block phage dissemination,” the authors wrote in their report.

Jackson highlighted the importance of the Cas12a3’s ability to halt protein production by cutting a specific region of tRNA known as the “tail.” This innovative approach represents a powerful method to prevent viral replication without damaging the host cell’s DNA, potentially leading to therapeutic breakthroughs.

The research team, including doctoral student Kadin Crosby and master’s student Bamidele Filani, played crucial roles in defining the functions of Cas12a3 and exploring its diagnostic capabilities. They designed synthetic reporters that mimic the tRNA acceptor stem and tail, thereby expanding the capacity of current CRISPR-based diagnostics for multiplexed RNA detection.

Jackson expressed optimism about the implications of their findings. “We think being able to stop an invading pathogen while leaving DNA unchanged could be a therapeutic breakthrough,” he said. The ongoing research aims to explore the extensive functional diversity within these bacterial defense mechanisms.

In summary, the discovery of RNA-mediated tRNA cleavage in the CRISPR-Cas systems reveals a rich functional diversity of antiviral defenses. The exploration of these mechanisms has the potential to expand the CRISPR toolbox significantly, with Cas12a3 emerging as a key player in multiplexed RNA detection.