A wealth of CRISPR-like gene-cutting enzymes found in microbes

By exploring the evolutionary origins of an enzyme used in the CRISPR genome editing system, researchers have uncovered more than a million other potential editors hidden in microbial genomes.

The study, published on September 9 in Science¹, found the new editing enzymes among a family of proteins called IscB. These proteins are believed to be the ancestors of the Cas9 enzyme, known as the molecular scissors of CRISPR. When editing the genome, Cas9 combines with an RNA extract that guides the enzyme to find and cut a specific DNA sequence. The technique’s reliance on RNA as a guiding system is one of the main reasons for its versatility and widespread use, as it allows researchers to easily target Cas9 to the region of the genome they wish to modify. .

The discovery of other RNA-targeted enzymes capable of cutting DNA could provide further tools for genome editing, according to study lead author Feng Zhang, molecular biologist at the Massachusetts Institute of Technology of Cambridge (MIT). “These programmable proteins are very useful, beyond basic biological interest,” he says. “And this RNA-guided DNA recognition mechanism is probably something that nature has independently created many times over.”

Although researchers have exploited it for genetic engineering, CRISPR is believed to be a microbial defense system that allows bacteria and other single-celled organisms called archaea to repel viruses and other genetic invaders by sending Cas9 to chew on their DNA. Computer studies have suggested that Cas9 likely evolved from proteins of the IscB family, which are encoded by transposons, or “jump genes,” which can jump to new locations in the genome. Until now, the function of IscB proteins has not been clear.

Zhang and his colleagues found that the DNA responsible for encoding IscB proteins is often located in close proximity to the DNA of a class of RNA molecules they have dubbed ωRNA. They also found that certain IscB proteins can cleave DNA at a site specified by an RNA sequence, much like Cas9 and its guide RNA.

The team then studied another family of proteins, called TnpB, which are said to be the ancestors of another enzyme associated with CRISPR that cuts DNA called Cas12. They found that some of these proteins could also cut DNA when guided by RNA.

Unexpected diversity

Database searches have revealed more than a million genes that may carry the code for TnpB proteins, and some organisms contain more than 100 copies of these genes, says Soumya Kannan, molecular biologist at MIT and co-first author of the study.

And the IscB genes have appeared not only in bacteria and archaea, but also in the light-collecting chloroplast inside an algal cell. This is the first time that such genome editing systems have been found in a eukaryote (the group of organisms whose cells contain nuclei, which includes all plants and animals) – a surprising result which suggests that ‘they are more widespread than previously thought. “Whenever I give a talk, people always ask me if we have seen CRISPR activity in a eukaryotic cell,” says Zhang. “Now I can finally say ‘yes’.”

In nature, these genes could perform a variety of functions, including defending or regulating the expression of other genes. And in the lab, the discovery could give rise to a treasure trove of editing tools. Zhang and his team found that the IscB could be used to cut human DNA, albeit with lower efficiency than the popular CRISPR-Cas9 system. But Zhang says the IscB system could be improved, and notes that the small size of the IscB protein could make it easier to work with some applications.

For geneticist Gaetan Burgio of the Australian National University of Canberra, the real beauty of the study is its contribution to understanding evolution – and ultimately attributing possible function to such a large group of proteins. and rumored that the IscB. “It’s absolutely fascinating,” he says. “It fills an important gap: we didn’t really know how these CRISPR systems came to be CRISPR. “