Ancient RNA-guided systems found in viruses could expand gene editing

Researchers at MIT's McGovern Institute and the Broad Institute, led by Feng Zhang, have identified a family of proteins they call TIGR-Tas (Tandem Interspaced Guide RNA), described online February 27 in the journal Science. The team found more than 20,000 different Tas proteins, most of them in viruses that infect bacteria. Separately, another group has described a different ancient system called VIPR (Viral Interference Programmable Repeat), reported in a paper posted to PubMed Central, which they argue is an ancestor of the RNA-guided proteins at the core of CRISPR itself.

Both systems work on the same basic principle as CRISPR-Cas9, the bacterial immune system that scientists adapted into today's most widely used gene-editing tool. In CRISPR-Cas9, a short RNA guide directs the Cas9 enzyme to a matching DNA sequence, which Cas9 then cuts; that guide RNA is built from fragments of virus DNA that bacteria store in their own genome as a kind of memory bank, so that when the same virus attacks again, the bacteria can recognize and destroy it. TIGR and VIPR are not variations of Cas9 — they use their own distinct proteins and RNA structures — but they perform a similar job of using RNA to find and act on a specific DNA site.

The Zhang lab found TIGR by searching hundreds of millions of proteins for a structural feature that lets Cas9 bind its RNA guide, then repeating the search from a more distantly related protein to widen the net. The results were so varied that the team used a protein language model, a type of artificial intelligence trained on protein structures, to sort them into likely evolutionary groups. One group stood out because its genes carried the kind of repetitive sequences seen in CRISPR arrays; these turned out to be the TIGR-Tas systems.

Each Tas protein has separate working parts, or modules: one binds the RNA guide, and in some proteins, an adjacent segment cuts the DNA. Because these parts are modular, researchers say they may be able to swap in new functions onto natural Tas proteins to build customized tools. The team showed that several Tas proteins can be programmed to cut targeted DNA sequences in human cells.

Two features may make TIGR-Tas particularly useful. CRISPR systems can only cut DNA next to short flanking sequences called PAMs (protospacer adjacent motifs); TIGR proteins have no such requirement, meaning, according to the researchers, that any site in the genome should in principle be reachable. The system also uses what one researcher called a "dual-guide" arrangement, interacting with both strands of the DNA double helix to lock onto its target. On top of that, Tas proteins are about a quarter of the size of Cas9 on average, which matters because smaller proteins are easier to package and deliver into cells.

VIPR was found by searching for structural features shared by a family of ancient CRISPR-associated proteins called RAMPs, rather than by starting from Cas9 directly. The resulting Vipr protein is simpler than the RAMPs found in modern CRISPR systems, and it turns up mostly inside bacteriophages and archaeal viruses rather than in bacterial genomes themselves. Researchers found that Vipr's genetic locus is minimal, typically just two genes, compared to the many genes packed into a CRISPR locus, and they place Vipr on an evolutionary branch that splits off before CRISPR's own RAMP proteins.

The RNA guide used by VIPR, called a vrRNA, does not match its DNA target the way CRISPR guides do. A CRISPR guide RNA pairs continuously, base by base, with its target. The vrRNA instead reads its target using a repeating pattern of a fixed three-letter motif alternating with two variable bases, skipping one base between each pairing site — a pattern the researchers call a "noncontiguous code." They confirmed this rule, called the 1-nucleotide skip rule, using computational search, biochemical binding tests, and a reporter-gene silencing experiment in bacteria, and found that disrupting the RNA's key motifs abolished the effect.

The researchers tested VIPR's function by using it to repress a reporter gene in bacteria, similar to a gene-silencing method used with disabled versions of Cas9, and found it worked on either DNA strand rather than being limited to one, unlike that Cas9-based method. Because natural VIPR-linked RNA targets tended to match sequences from competing viruses, the researchers propose that VIPR's biological role is helping viruses interfere with rival viruses infecting the same bacterial cell.

CRISPR-Cas9 is already used in research and in clinical trials for conditions including cystic fibrosis, hemophilia, and sickle cell disease, and it has been used to edit large viral genomes such as adenovirus and herpes simplex virus for vaccine and gene-therapy applications. But delivering CRISPR components into the body remains a practical obstacle, and existing PAM-sequence restrictions limit which DNA sites can be targeted. The small size of Tas proteins is presented by the MIT and Broad researchers as a potential advantage for therapeutic delivery, and the lack of a PAM requirement as a potential advantage for targeting flexibility.

Both discoveries are early. The Zhang lab is still investigating the natural role TIGR systems play inside viruses and has so far determined the molecular structure of only one working Tas protein, which they plan to use as a guide for making the system more efficient; they also note possible connections between TIGR-Tas and RNA-processing proteins in human cells that they say merits further study. The VIPR findings so far describe function only in a bacterial and bacteriophage context — gene silencing in engineered E. coli — with no demonstration reported yet of use as a human gene-editing tool. Neither system has undergone the kind of clinical testing that CRISPR-Cas9 has, and general CRISPR-based reviews caution that off-target effects, delivery to specific tissues, and other safety and regulatory questions still require rigorous preclinical and clinical study before any RNA-guided tool reaches patients.