New Programmable Gene Editing Proteins Found Outside CRISPR Systems

Over the past decade, scientists have adapted the CRISPR systems of microbes to gene editing technology, a precise and programmable system for modifying DNA. Today, scientists at the McGovern Institute at MIT and the Broad Institute at MIT and Harvard have discovered a new class of programmable DNA modification systems called OMEGA (Obligate Mobile Element Guided Activity), which may naturally be involved in the shuffling of small pieces of DNA into bacterial genomes.

These ancient DNA-cutting enzymes are guided to their targets by small pieces of RNA. Although they originate from bacteria, they have now been designed to function in human cells, which suggests that they could be useful in the development of gene-editing therapies, especially since they are small ( about 30% the size of Cas9), which makes them easier to administer. cells than larger enzymes. The discovery, reported in the newspaper Science, provides evidence that natural RNA-guided enzymes are among the most abundant proteins on earth, pointing to a vast new field of biology that is poised to lead the next revolution in genome editing technology .

The research was led by Feng Zhang, McGovern researcher, James and Patricia Poitras professor of neuroscience at MIT, researcher at Howard Hughes Medical Institute and senior fellow of the Broad Institute. Zhang’s team is exploring natural diversity in search of new molecular systems that can be rationally programmed.

“We are very excited to discover these widespread programmable enzymes, which have always been lurking under our noses,” said Zhang. “These results suggest the tantalizing possibility that there are many other programmable systems waiting to be discovered and developed as useful technologies.”

Programmable enzymes, especially those that use an RNA guide, can be quickly adapted for different uses. For example, CRISPR enzymes naturally use an RNA guide to target viral invaders, but biologists can point Cas9 to any target by generating their own RNA guide. “It’s so easy to just change a guidance sequence and set a new target,” says Soumya Kannan, graduate student and co-first author of the article. “So one of the general questions we’re interested in is trying to see if other natural systems use this same type of mechanism.”

The first clues that OMEGA proteins could be directed by RNA came from genes for proteins called IscB. IscBs are not involved in CRISPR immunity and were not known to associate with RNA, but they looked like small DNA-cutting enzymes. The team found that each IscB had a small encoded RNA nearby, and it directed the IscB enzymes to cut specific DNA sequences. They named these RNAs “ωRNA”.

The team’s experiments showed that two other classes of small proteins known as IsrB and TnpB, one of the most abundant genes in bacteria, also use RNAs that serve as guides to direct cleavage. DNA.

IscB, IsrB, and TnpB are found in mobile genetic elements called transposons. Graduate student Han Altae-Tran, co-first author of the article, explains that each time these transposons move, they create a new guide RNA, allowing the enzyme they code to cleave elsewhere.

It’s not clear how bacteria benefit from this genomic shuffling, or if they do at all. Transposons are often thought of as selfish fragments of DNA, concerned only with their own mobility and conservation, Kannan explains. But if hosts can “co-opt” these systems and reuse them, hosts can acquire new capabilities, such as with CRISPR systems that confer adaptive immunity.

IscBs and TnpBs appear to be the predecessors of CRISPR Cas9 and Cas12 systems. The team suspects that they, along with IsrB, have likely also spawned other RNA-guided enzymes – and they’re eager to find them. They are curious about the range of functions that could be performed in nature by RNA-guided enzymes, says Kannan, and evolution suspects probably already took advantage of OMEGA enzymes like IscBs and TnpBs to solve problems that biologists wish to resolve.

“A lot of things we’ve been thinking about may already exist naturally to some extent,” says Altae-Tran. “Natural versions of these types of systems could be a good place to start to adapt to this particular task.”

The team is also interested in tracing the evolution of RNA-guided systems further into the past. “Finding all of these new systems highlights the evolution of RNA-guided systems, but we don’t know where the RNA-guided activity itself comes from,” says Altae-Tran. Understanding these origins, he says, could pave the way for the development of even more classes of programmable tools.