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UC Berkeley Team Uncovers New CRISPR/Cas Gene-Editing Systems

NEW YORK (GenomeWeb) – Researchers from the University of California, Berkeley this week reported on the discovery of two new CRISPR/Cas systems in a variety of uncultivated microbes, opening the door for the development of new versions of the genome-editing technology.

The team also uncovered the existence of genes encoding Cas9 — the enzyme that cleaves double-stranded DNA at specific locations during the CRISPR process — in archaea, marking the first evidence that the CRISPR system exists in these prokaryotes.

Since CRISPR/Cas9 was originally discovered, it was presumed to exist only in the bacterial domain. As a result, current CRISPR/Cas technologies are derived from cultured bacteria, "leaving untapped the vast majority of enzymes from organisms that have not been cultured," the scientists wrote in a paper appearing in Nature.

Aiming to explore the possibility of new CRISPR/Cas systems, the scientists used genome-resolved metagenomics to study microbial communities found in groundwater, soil, mine drainage biofilms, the human infant gut, and other environments, specifically targeting large uncharacterized genes proximal to a CRISPR array and the universal CRISPR integrase cas-1.

Among the 155 million protein-encoding genes that were analyzed, ones that encode Cas9 were found in the genomes of the acid-mine dwelling nanoarchaea ARMAN-1 and ARMAN-4, expanding the presence of Cas9-containing CRISPR systems to another domain of life.

The investigators also discovered in uncultured bacteria two new CRISPR/Cas systems — dubbed CRISPR/CasX and CRISPR/CasY — that are among the most compact CRISPR/Cas loci identified to date and are found exclusively in metagenomic datasets. "The small number of proteins that are required for interference and their relatively short length make these systems especially valuable for the development of genome-editing tools," the scientists wrote.

Some of these compact loci were identified in organisms with very small genomes, they noted. As a result, they wrote, "these organisms likely depend on other community members for basic metabolic requirements and thus have remained largely outside the scope of traditional cultivation-based methods."

The activity of both the CRISPR/CasX and CRISPR/CasY systems was validated in Escherichia coli in the lab of UC Berkeley scientist and CRISPR/Cas9 pioneer Jennifer Doudna, who co-authored the Nature paper.

Overall, the findings expand the repertoire of microbe-based biotechnologies, and demonstrate that metagenomic discoveries related to CRISPR-Cas systems are "not restricted to in silico observations, but can be introduced into an experimental setting where their activity can be analyzed," the researchers concluded.

"Given that virtually all environments where life exists can now be probed by metagenomic methods, we anticipate that the combined computational-experimental approach will greatly expand the diversity of known CRISPR-Cas systems, enabling new technologies for biological research and clinical applications."