Cas3, which shreds where Cas9 snips, has been used to chew up long stretches of DNA. Even better, it has done so with precision, a quality seldom associated powerful shredding tools. Combining exactitude and voracity, Cas3, as part of a minimal CRISPR system, has deleted genomic regions as large as 424 kb with high efficiency and speed.
This feat was achieved at the University of California, San Francisco (UCSF), where scientists led by Joseph Bondy-Denomy, PhD, selected and modified the CRISPR-Cas3 system used by the bacterium Pseudomonas aeruginosa. This system, the scientists declared, qualifies as a “minimal” Type I-C CRISPR-Cas systems system. That is, it utilizes just three cas genes (cas5, cas8, and cas7) to produce the crRNA-guided Cascade surveillance complex that can recruit Cas3.
After the scientists repurposed the CRISPR-Cas3 system for genome editing, they evaluated its function in P. aeruginosa and three other microbes, Escherichia coli, Pseudomonas syringae, and Klebsiella pneumoniae. For example, the scientists tried in three applications: the single-step deletion of large virulence regions, multiplexed targeting, and plasmid curing.
Details of this work appeared in the journal Nature Methods, in an article titled, “A compact Cascade–Cas3 system for targeted genome engineering.”
“DNA cleavage guided by a single CRISPR RNA generated large deletions (7–424 kilobases) in Pseudomonas aeruginosa with near-100% efficiency, while Cas9 yielded small deletions and point mutations,” the article’s authors wrote. “Cas3 generated bidirectional deletions originating from the programmed site, which was exploited to reduce the P. aeruginosa genome by 837 kb (13.5%). Large deletion boundaries were efficiently specified by a homology-directed repair template during editing with Cascade–Cas3, but not Cas9.”
Gene editing for the development of new treatments, and for studying disease as well as normal function in humans and other organisms, may advance more quickly with a new tool for cutting larger pieces of DNA out of a cell’s genome, the UCSF scientists argued. A candidate for such a tool is the compact CRISPR-Cas3 system derived from P. aeruginosa.
Other CRISPR-Cas3 systems have been made to work in human and other mammalian cells, and that also should be achievable for the modified P. aeruginosa system, Bondy-Denomy said.
The already renowned CRISPR-Cas9 ensemble can be used to rapidly and precisely excise a small bit of DNA at a targeted site. Other methods can then be used to insert new DNA. But the new CRISPR-Cas3 system adapted by the UCSF scientists is different. The key enzyme in this system, Cas3, removes much longer stretches of DNA quickly and accurately.
“Cas3 is like Cas9 with a motor—after finding its specific DNA target, it runs on DNA and chews it up like a Pac-Man,” Bondy-Denomy explained. Unlike Cas9, when Cas3 binds to its precise DNA target it begins chewing up one strand of the double-stranded DNA in both directions, leaving a single strand exposed.
The deletions obtained in the UCSF experiments ranged in size, in many cases encompassing as many as 100 bacterial genes. The CRISPR-Cas3 mechanism should also allow for easier replacement of deleted DNA with a new DNA sequence, the researchers found.
“CRISPR-Cas3 is an especially promising tool for use in eukaryotic cells as it would facilitate the interrogation of large segments of noncoding DNA, much of which has unknown function,” the article’s authors added. “Additionally, it was recently shown that Cas9-generated ‘gene knockouts’ (i.e., small indels causing out-of-frame mutations) frequently encode pseudo-mRNAs that may produce protein products, necessitating methods for full gene removal.”
According to Bondy-Denomy, there has been no easy and reliable way to delete very large regions of DNA in bacteria for research or therapeutic purposes. “Now,” he suggested, one needn’t make “100 different small DNA deletions.” Instead, one may make just one deletion and ask, “What changed?”
Because bacteria and other types of cells are commonly used to produce small molecule or protein-based pharmaceuticals, CRISPR-Cas3 will enable biotechnology industry scientists to more easily remove potentially pathogenic or useless DNA from these cells, according to Bondy-Denomy.
“Large swathes of bacterial DNA are poorly understood, with unknown functions that in some cases are not necessary for survival,” Bondy-Denomy elaborated. “In addition, bacterial DNA contains large stretches of DNA imported from other sources, which can cause disease in the bacterium’s human host, or divert bacterial metabolism.”
CRISPR-Cas3 also should also allow entire genes to be inserted into the genome in industrial, agricultural, or even in human gene therapy applications.
In the new CRISPR-Cas3 study, by manipulating the sequences of DNA provided to the bacteria for repairing the deletions, the researchers were able to precisely set the boundaries of these large DNA repairs, something they were unable to accomplish with CRISPR-Cas9.
Bondy-Denomy previously discovered anti-CRISPR strategies that phage evolved to fight back against bacteria, and these might prove useful for stopping the gene editing reactions driven by Cas enzymes used as human therapeutics before side effects arise, or in using phage to remove unwanted bacteria that have populated the gut, he said. Apart from E. coli and a couple of other species, relatively little is known about the 1,000 or so bacterial species that normally reside there.
“Non-model microbes,” Bondy-Denomy concluded, “have largely been left behind in the genetics world, and there is a huge need for new tools to study them.”