CRISPR Meets Pac-Man: New DNA Cut-and-Paste Tool Enables Bigger Gene Edits

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, according to a new study by UC San Francisco scientists.

Publication of the UCSF study on Oct. 19, 2020 in the journal Nature Methods comes less than two weeks after two researchers who first used the genetic scissors known as CRISPR-Cas9 were selected to receive this year’s Nobel Prize in Chemistry.

Though now employed as a research tool in laboratories around the world, CRISPR evolved eons ago in bacteria as a means to fight their ancient nemesis, a whole host of viruses known as bacteriophages. When bacteria encounter a phage, they incorporate a bit of the viral DNA into their own DNA, and it then serves as a template to make RNA that binds to the corresponding viral DNA in the phage itself. The CRISPR enzymes then target, disable and kill the phage.

In his latest work exploring this ancient and strange arms race, principal investigator Joseph Bondy-Denomy, PhD, associate professor in the UCSF Department of Microbiology and Immunology, joined scientists Bálint Csörgő, PhD, and Lina León to develop and test a new CRISPR tool.

The already renowned CRISPR-Cas9 ensemble is like a molecular chisel that 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 employs a different bacterial immune system. The key enzyme in this system, Cas3, acts more like a molecular wood chipper to remove 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 said.

This new capability to delete or replace long stretches of DNA will enable researchers to more efficiently assess the importance of genomic regions that contain DNA sequences of indeterminate function, according to Bondy-Denomy, an important consideration in understanding humans and the pathogens that plague them.

“Previously, there was no easy and reliable way to delete very large regions of DNA in bacteria for research or therapeutic purposes,” he said. “Now, instead of making 100 different small DNA deletions we can just make 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 said. “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, Bondy-Denomy said.

The UCSF researchers selected and modified the CRISPR-Cas3 system used by the bacterium Pseudomonas aeruginosa, and demonstrated in this species and in three others, including bacteria that cause disease in humans and plants, that their more compact version functions well to remove selected DNA in all four species. 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.

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