Smaller life forms have the smallest CRISPR system that works

An old group of microbes containing some of the smallest life forms on Earth also has the smallest gene editing mechanism CRISPR discovered to date.

Many archaeas like these have CRISPR systems to protect themselves from viruses. The smallest CRISPR system found to date, Cas14, has been found in the genome of one of these Archaea, which scientists have so far not been able to cultivate in the laboratory.

The peewee protein machine, called Cas14, is only one-third the size of the Cas9 protein, the commercial aspect of the revolutionary CRISPR-Cas9 gene editing tool. While Cas9 was isolated from bacteria, Cas14 was found in the genome of a group of Archaea – a primitive parent of bacteria – that contains some of the smaller cells and smaller known genomes.

Cas9 and other protein cases are part of a defense system developed by microbes to protect themselves from viruses. All are targeted enzymes that search and bind very selectively to a specific DNA or RNA sequence – in microbes, those that correspond to sequences stored in its CRISPR memory banks after previous viral infections – and then cut DNA or RNA to disable the new invader.

Like Cas9, Cas14 has potential as a biotechnology tool. Because of its small size, Cas14 could be useful for gene editing in small cells or in some viruses. But with its single-stranded DNA cleavage activity, it is more likely to improve the rapid CRISPR diagnostic systems currently 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 graduate student at the University of Berkeley and first author of an article reporting this discovery. "Cas12 is really good for double-stranded DNA recognition, Cas13 is really good for recognition of single-stranded RNA and now Cas14 completes the set: it's really good for recognition of single stranded DNA. "

Cas14 is similar to Cas12 and Cas13 in that, after being bound to its target DNA sequence, it begins to indiscriminately cut all the single-stranded DNA inside of it. 39, a cell. On the other hand, Cas9 binds and cuts only the targeted DNA.

The free cut of the DNA is a possible disadvantage in therapy, but a considerable advantage in diagnosis. The Cas14 protein can be coupled to a fluorescent label attached to a single-stranded DNA fragment. When Cas14 binds to its target DNA sequence – a cancer gene or an infectious bacterium gene – and begins to cut DNA, it also cleaves the DNA bound to the marker, generating a fluorescent signal.

"Cas14 targets single-stranded DNA in a much more specific way than Cas12," added Janice Chen, a colleague from Harrington, who has just obtained her PhD. from UC Berkeley. "It was a really unexpected discovery. Because it's so small, we hardly thought it could work, but in reality, it's extremely specific, which also makes it a very powerful addition to the diagnostic toolbox. "

Harrington, Chen and their colleagues, including Jennifer Doudna, inventor of CRISPR-Cas9, a professor of molecular and cellular biology and chemistry at Berkeley, have adapted Cas14 to work with their diagnostic system, called DETECTR, which now uses Cas12 and Cas13 to quickly detect the presence of infectious organisms and genetic mutations. Harrington, Doudna and Chen are co-founders of a company, Mammoth Biosciences, which markets DETECTR.

The discovery will be reported online on October 18 before publication in the newspaper. Science. Doudna is a researcher at the Howard Hughes Medical Institute, co-director of the Innovative Genomics Institute and a faculty scientist at the Lawrence Berkeley National Laboratory. Banfield is responsible for microbiology for IGI and a subsidiary of Berkeley Lab.

Mining metagenomes

The Cas14 protein was discovered by co-first authors Harrington and David Burstein, now a professor at the Tel Aviv University in Israel, while they were looking for variant cases in a database. Microbial genome data created over the past 15 years by their colleagues at the UC Berkeley – team led by Jill Banfield, professor of Earth Sciences and Planets and Environmental Sciences, Policy and Economics. of the management. Genomes, numbered in tens of thousands, were obtained by metagenomic sequencing of all DNA in samples from various exotic environments. Cas14 was found in the genome of Archaea sequenced from groundwater samples from a toxic depollution site located in Rifle, Colorado.

The smallest known CRISPR gene editing system was found in a database of all microbial genomes sequenced from the ground up in a toxic cleaning site located in Rifle, Colorado. (Image of Iris Burstein)

Two years ago, Harrington and Burstein discovered other small proteins Cas, CasX and CasY, extracting the metagenomic database.

Cas14 has a size equal to half the size – between 400 and 700 amino acids – of CasX and is smaller than all other known Cas systems, ranging in length from 950 to 1400 amino acids.

"By chance, we found these very small proteins that other people simply throw away because they do not look like CRISPR systems previously known. They are too small, "Harrington said. "We decided, damn it, let's try." We tested it and we were shocked to find that it was real functional systems. "

Finding the Cas14 gene in the database was just the beginning. To date, most Cas proteins have been found in bacteria and therefore work well in standard laboratory bacteria. E. coli. But Cas14 comes from Archaea – and from a group of the smaller Archaea, called DPANN. All Cas proteins contain RNA bits for targeting and binding, but Cas14 will not work with CRISPR-Cas9 RNAs. The team also had to extract from the database the two ARNs that must be present for Cas14 to function.

In addition, DPANN Archaea can not be grown in the lab – they appear to be parasitic or somehow depend on other larger Archaea – so the researchers had to create the right environment in a test tube.

The archaic and eukaryotic branches (animals and plants) of the tree of life, showing the position of the DPANN group from which Cas14 was isolated. The bacteria, not shown, are along the line that stretches from the top of the image.

Consistent with its origin in a more primitive microbe, the lightened Cas14 seems to be a more primitive version of the larger and more complex Cas9 and Cas12 proteins, Harrington said, noting that molecules have evolved to become more specialized. The researchers hope to learn from these primitive case proteins, which are the essential components of the enzyme case, in order to be able to design the most compact and elegant gene cutters possible.

Harrington noted that metagenomic extraction has developed various versions of Cas14 that could prove to be useful biotechnological tools. "An amazing thing … is the diversity of these systems," he said. "We have described more than 40 new CRISPR-Cas14 systems and eight different subtypes. This opens the door to an investigation of these new CRISPR systems. "

The co-authors with Harrington, Burstein, Chen, Doudna and Banfield are Enbo Ma, Isaac Witte, Joshua Cofsky of UC Berkeley and David Paez-Espino and Nikos Kyrpides of the Common Genome Institute of the Department of Energy in Walnut Creek , in California. The metagenomic databases extracted by the group are available in JGI's Integrated Microbial Genome and Microbiome Integrated System (IMG / M), the largest collection of microbial genes, currently growing by 55 billion.

The work was funded by the National Science Foundation, the US Department of Energy, the Innovative Genomics Institute and the Paul Allen Institute.

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