Image: Many Archaea like these have CRISPR systems to protect themselves from attacking viruses. The smallest CRISPR system found to date, Cas14, was found in the genome of one such Archaea, which scienstists have so far been unable to grow in the lab.
An ancient group of microbes that contains some of the smallest life forms on Earth also has the smallest CRISPR gene-editing machinery discovered to date.
The peewee protein machinery, dubbed Cas14, is related to but one-third the size of the Cas9 protein, the business end of the revolutionary gene-editing tool CRISPR-Cas9. While Cas9 was isolated from bacteria, Cas14 was found in the genome of a group of Archaea – a primitive relative of bacteria – that contains some of the smallest cells and smallest genomes known.
Cas9 and other Cas proteins are part of a defense system evolved by microbes to protect themselves from viruses. All are targeted enzymes that seek out and bind very selectively to a specific DNA or RNA sequence – in microbes, those that match sequences stored in its CRISPR memory banks after earlier viral infections – and then cuts the DNA or RNA to disable the new invader.
Like Cas9, Cas14 has potential as a biotech tool. Because of its small size, Cas14 could be useful in editing genes in small cells or in some viruses. But with its single-stranded DNA cutting activity, it is more likely to improve rapid CRISPR diagnostic systems now under development for infectious diseases, genetic mutations and cancer.
“For molecular diagnostics, you want to be able to target double-stranded DNA, single-stranded DNA and RNA,” said Lucas Harrington, a UC Berkeley graduate student and first author of a paper reporting the discovery. “Cas12 is really good at double-stranded DNA recognition, Cas13 is really good at single-stranded RNA recognition and now Cas14 completes the set: it is really good at single-stranded DNA recognition.”
Cas14 is similar to Cas12 and Cas13 in that, after binding to its target DNA sequence, it begins indiscriminately cutting all single-stranded DNA inside a cell. Cas9, in contrast, binds and cuts only the targeted DNA.
The wanton cutting of DNA is a possible disadvantage in therapy, but a great advantage in diagnostics. The Cas14 protein can be paired with a fluorescent marker attached to a piece of single-stranded DNA. When Cas14 binds to its target DNA sequence – a cancer gene or a gene in infectious bacteria – and starts cutting DNA, it will also cut the DNA linked with the marker, generating a fluorescent signal.
“Cas14 targets single-stranded DNA in a much more specific way than Cas12 does,” added Harrington’s colleague, Janice Chen, who recently received her Ph.D. from UC Berkeley. “That was a really unexpected finding. Because it is so small, we barely thought it could work, but actually it is super-specific, which makes it also a really powerful addition to the diagnostic toolbox.”