Professor Wendy Harwood poses for a photograph in a plant breeding incubator room with barley plants that have undergone gene editing at the John Innes Centre in Norwich, Britain, May 25, 2016. (REUTERS/File Photo/Stuart McDill)
SAN FRANCISCO, July 12 (Xinhua) -- While a gene-editing technology is based on a tactic bacteria developed to protect themselves from viruses, new research shows that the countermeasure viruses can be used to improve the same technology as a gene-therapy tool.
The finding, reported online this week in the journal Science Advances, could decrease off-target gene editing that could cause unwanted side effects.
The technology in the study by researchers from the University of California, Berkeley, and the University of California, San Francisco, is known as clustered regularly interspaced short palindromic repeats associated protein 9, or CRISPR-Cas9. And the countermeasure viruses they came up with are inhibitory proteins referred to as anti-CRISPRs.
One particular anti-CRISPR protein, called AcrIIA4, according to the researchers, reduced by four-fold the off-target effects of a CRISPR-Cas9 molecule that uses a guide ribonucleic acid (RNA) to find, snip and replace the mutated hemoglobin gene responsible for sickle cell disease. It does this without significantly reducing the desired on-target gene-editing.
"Unexpected mutations can arise as a result of off-target gene editing, but our paper -- like many others -- shows that off-target effects can be modulated and it is not as serious as people might think," UC Berkeley postdoctoral fellow Jiyung Jenny Shin, from the lab of Jacob Corn at the Innovative Genomics Institute (IGI) and one of three first authors of the paper, was quoted as saying in a news release.
In experiments on human cells in culture, Shin found that delivering CRISPR-Cas9 and then the anti-CRISPR protein was the most effective way to reduce off-target effects. The protein mimics deoxyribonucleic acid (DNA), glomming onto Cas9, the enzyme that actually cuts the double-stranded DNA, and preventing further cutting. "Even after six hours of effective CRISPR, inserting anti-CRISPR decreases off-target effects by more than two-fold compared to on-target effects," she said. "Therapeutically, you could treat a patient with CRISPR first, and then treat with anti-CRISPR at a later time and decrease off-target effects."
The researcher who discovered AcrIIA4, Joseph Bondy-Denomy of UC San Francisco, foresees these anti-CRISPR proteins becoming a standard part of CRISPR gene therapy, given along with CRISPR-Cas9 to disable gene editing after a fixed period of time to prevent random off-target cutting. "This Cas9 inhibitor could be encoded on the same piece of DNA as Cas9, for example, precisely timed to turn Cas9 off after the gene editing is done, instead of letting Cas9 linger in the cell and risk off-target effects," said Bondy-Denomy, who is a co-author of the paper.
"Jenny's data suggests that there is an ideal time window for letting Cas9 do its job and then turning it off after that amount of time has passed," Bondy-Denomy said. "We can actually use the anti-CRISPR proteins as tools to figure out what that time window is, that is, for any one cell type with any one guide RNA sequence, how long we want Cas9 to be active in the cell."
The CRISPR inhibitor targets a spot on the Cas9 protein that is essential for Cas9's function.
Previous research suggests that CRISPR-Cas9 constantly feints with the cell's DNA repair system: as the enzyme cuts at its target site, the cell repairs the DNA, and CRISPR-Cas9 cuts again, repeating this vicious cycle until a mutation arises in the DNA that prevents enzyme binding, at which point the CRISPR-Cas9 molecule moves on to find another binding site.
The current work suggests that adding an anti-CRISPR after Cas9 has edited a target gene would prevent unintended damage to other portions of a genome.
"The ability to turn Cas9 gene editing off is just as important as the ability to turn it on," noted Corn, scientific director for biomedicine of the IGI and a UC Berkeley assistant adjunct professor of molecular and cell biology. "Imagine if you had an electric razor with no off-switch! For eventual therapeutic applications, it is critical to be able to precisely control when and where gene editing is active. The anti-CRISPR proteins offer opportunities to completely turn off Cas9 as well as fine-tune its activity."
