This DNA-mimicking protein can make gene editing more precise and safe

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Scientists have discovered a virus-made protein that can block the powerful gene-editing tool CRISPR-Cas9 from cutting DNA. The protein allows researchers to better control CRISPR so that it doesn’t snip unintended pieces of genetic code. In the future, the technique could be used to make gene editing more precise — and safe.

The protein, called AcrIIA4, switches gene editing off by mimicking DNA: it basically acts like a decoy, fooling CRISPR’s molecular scissors into thinking they’re cutting actual DNA. Scientists at several institutions — including CRISPR pioneer Jennifer Doudna at the University of California, Berkeley — showed that the protein could reduce undesired gene changes in human blood cells. The findings were published today in the journal Science Advances.

CRISPR-Cas9 is based on a defense mechanism bacteria use to ward off viruses — cutting off bits of viral DNA. Scientists have engineered that naturally occurring mechanism to reorder bits and pieces of the genetic code, and have used it to create unusually muscular beagles, for instance, and mosquitoes that don’t transmit malaria. Recently, scientists have discovered that viruses have their own anti-CRISPR counter-measures: they produce proteins that can turn off that deadly genetic snipping. And now, researchers are trying to use this molecular “arms race” to better control our own tweaked CRISPR systems.

Even though gene-editing tools like CRISPR-Cas9 are very precise, they sometimes snip pieces of DNA they weren’t programmed to cut. These off-target cuts can be dangerous, and scientists have been trying to find ways to prevent them. “It’s very easy to make the cuts with Cas9, but really controlling the nature of those cuts is crucial,” says Dane Hazelbaker, a CRISPR research scientist at the Broad Institute, who was not involved in the study.

Today’s research shows how one of these viral anti-CRISPR proteins works, and how it affects a commonly used type of gene-editing tool. The researchers found that the protein AcrIIA4 mimics DNA so that it can bind to the Cas9 enzyme, blocking it from attaching to actual DNA and cutting it. The scientists then ran several experiments on human blood cells, to understand how to use the protein to prevent unintended, off-target cuts. They added the anti-CRISPR protein to the cells before using CRISPR-Cas9, but that switched CRISPR’s molecular scalpel off completely — blocking both the intended and unintended editing. Then, they added the anti-CRISPR protein and the Cas9 together, and that, too, decreased both wanted and unwanted edits. Finally, the researchers added AcrIIA4 a few hours after adding the Cas9; that prevented CRISPR from cutting DNA at the wrong sites, while still allowing time for cutting at the right sites. (The intended editing was decreased just a little.)


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How the protein AcrIIA4 affects Cas9.
Illustration: Shin et al. Sci. Adv. 2017;3: e1701620

That’s probably because CRISPR-Cas9 likely cuts the DNA it’s programmed to target before going awry and snipping undesired pieces of genetic code. Or it could be that the off-target cutting just happens more slowly, says study co-author Jacob Corn, an assistant adjunct professor of biochemistry, biophysics, and structural biology at UC Berkeley. “There is this timing argument,” he says. Corn and his colleagues were able to use this time lag to make CRISPR more precise.

It’s like having a surgeon “who’s scissors-happy, and that would be Cas9,” Hazelbaker says. You want the surgeon to do what he has to do — say, remove the appendix — but you don’t want him to also remove any of the nearby organs, like your gallbladder. The anti-CRISPR protein basically “yanks the scissors out of his hands before he can cut anything else,” Hazelbaker says.

The technique is incredibly important to make sure gene editing is done safely. In the case of diseases like sickle cell anemia, for instance, scientists would like to take blood cells from a patient, edit them using CRISPR, and then inject the cells back into the patient. But you’d want to make sure that CRISPR only cuts the bits of DNA it’s supposed to, and using anti-CRISPR proteins could help scientists do that. “In a therapeutic context, your tolerance for off-targets is going to be next to zero,” says Blake Wiedenheft, an assistant professor in the department of immunology and infectious diseases at Montana State University, who did not take part in the study.

There’s lots of work that needs to be done before AcrIIA4 — or other anti-CRISPR proteins — can be actually used to help cure people. “This not something you can go down to the drug store and get,” Corn says. Scientists need to figure out if this protein is completely safe, and whether it works in other human cells like stem cells, for instance. But it shows just how much potential is hidden inside these anti-CRISPR systems naturally devised by viruses. Scientists should closely analyze more of these proteins, to see if they can find other ways to better control CRISPR. “There’s a whole zoo of anti-CRISPR proteins,” Corn says.

Wiedenheft agrees, and says the potential is endless. “That’s where the hot spot of innovation exists.”

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