Identifying an RNA-guided system with promising gene-editing potential

An ancient RNA-guided system with potential to expand gene-editing capabilities has been identified.

Researchers at MIT’s McGovern Institute and the Broad Institute of MIT and Harvard (both MA, USA) have uncovered an ancient RNA-guided system that can target specific sites on DNA. The system has the potential to expand the genome-editing toolbox, rivaling current predominant genome-editing techniques due to its versatility and compact nature. This work could have significant implications for therapeutics development.

The researchers set out to investigate RNA-guided DNA-targeting proteins in nature, taking advantage of its diversity, to find novel programmable systems. “Being RNA-guided makes [the system] relatively easy to reprogram, because we know how RNA binds to other DNA or other RNA,” senior author Feng Zhang (both institutions) explained.

The team first focused on a structural feature of the CRISPR Cas9 protein that is key to its success as a genome-editing tool; this structural feature is responsible for binding to the enzyme’s RNA guide. The CRISPR structural domain served as a starting point for a series of iterative structural and sequence homology-based mining to find related RNA-guided proteins.

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As their search continued, the proteins identified were more and more distantly related to the initial input, prompting the researchers to turn to AI for help. “When you are doing iterative, deep mining, the resulting hits can be so diverse that they are difficult to analyze using standard phylogenetic methods, which rely on conserved sequence,” commented Guilhem Faure, first author of the paper and a computational biologist in Zhang’s lab.

Using a combination of homology-based mining and a protein large language model, the researchers grouped the proteins identified based on their likely evolutionary relationships. They were most intrigued by a group called the TIGR (Tandem Interspaced Guide RNA)-Tas system because they were encoded by genes with regularly spaced repetitive sequences reminiscent of CRISPR systems. They discovered over 20,000 different Tas proteins, predominantly from phage and parasitic bacteria, that encode an RNA guide that interacts with the RNA-binding domain of the protein itself.

The team went on to demonstrate how some Tas proteins can be programmed to edit DNA in human cells, revealing key characteristics of the system that make it a promising gene-editing candidate. TIGR systems have distinct functional modules that can act on the targeted DNA, which could allow researchers to swap useful features into natural Tas proteins during tool development. Additionally, unlike CRISPR, TIGR Tas proteins do not need DNA to be flanked by specific motifs to be directed to those segments, meaning any site in the genome should be fair game for editing. Further, TIGR systems have a dual-guide system, which relies on both sides of the DNA double helix to direct them to the appropriate sequences. And, finally, Tas proteins are a quarter of the size of the Cas9 protein on average, making gene editing tools easier to deliver for therapeutic purposes.

Next, the team is investigating TIGR’s natural role in viruses in the hopes of better understanding the system and how it can be adapted for research and therapeutics.