Joe Bondy-Denomy, a graduate student in the early 2010s, tried to infect bacteria with viruses that, on paper, shouldn’t have stood a chance. He knew that these viruses, or phages, were susceptible to CRISPR–Cas, the bacterial defense system that scientists have harnessed as a powerful tool for gene editing. And in most cases, he was right: the CRISPR machinery chopped the incoming phages into bits. But in a few instances, against the odds, the intruders survived. Bondy-Denomy—together with Davidson, microbiologist Karen Maxwell and fellow graduate student April Pawluk—had stumbled onto tools now known as anti-CRISPRs. These proteins serve as the rocks to CRISPR’s molecular scissors. And soon, they were popping up everywhere: more than 50 anti-CRISPR proteins have now been characterized, each with its own means of blocking the cut-and-paste action of CRISPR systems.
The expansive roster opens up many questions about the archaic arms race between bacteria and the phages that prey on them. But it also provides scientists with a toolkit for keeping gene editing in check. Some are using these proteins as switches to more finely control the activity of CRISPR systems in gene-editing applications for biotechnology or medicine. Others are testing whether they, or other CRISPR-stopping molecules, could serve as biosecurity counter-measures of last resort, capable of reining in some genome-edited bioweapon or out-of-control gene drive.
Yet, despite a growing number of proposed applications and proof-of-concept experiments in the laboratory, researchers have yet to pin down the therapeutic potential of these anti-CRISPR systems. Jennifer Doudna, a biochemist at the University of California, Berkeley, and one of the pioneers of CRISPR gene editing, voices a question that she says is on everyone’s lips: “How do you actually use these in a way that will provide meaningful control?”
“All Hell Breaks Loose”
In December 2016, Pawluk, still working in Davidson’s lab, and Bondy-Denomy, leading his own independent research group, each identified inhibitors to the Cas9 enzyme. This time, researchers around the world seized on the findings. “Like everything else in the CRISPR world, the thin edge of the wedge comes in, and the next thing you know all hell breaks loose,” says Erik Sontheimer, a molecular biologist at the University of Massachusetts Medical School in Worcester and a co-author on Pawluk’s paper.
In less than three months, structural biologists at the Har bin Institute of Technology in China had deciphered the molecular mechanism by which one of Bondy-Denomy’s anti-CRISPR proteins, called AcrIIA4, shut off Cas9 activity (see ‘CRISPR correctives’). A few months later, Doudna, working with Bondy-Denomy and biochemist Jacob Corn, now at the Swiss Federal Institute of Technology in Zürich, offered the first demonstration that anti-CRISPRs had practical value: they showed that delivering AcrIIA4 into human cells, either alongside or right after introducing Cas9, could halt gene-editing activity in its tracks.
In 2019, research teams in Germany, Japan and the United States independently attempted to use the proteins in tandem with small regulatory molecules called microRNAs to bring about tissue-specific editing. The US team, led by Sontheimer, even showed that the approach could work in mice—theirs is the only published study so far to demonstrate that anti-CRISPR proteins can work in a living animal, and not just cells. Although the paper focused on liver-directed editing, the platform is “plug and play”, says Sontheimer: any organs that produce a unique microRNA at high expression levels could be targeted in this way, provided that the anti-CRISPR proteins don’t trigger unwanted immune effects.
Not Immune to Challenges
Because of previous exposure to microbes harbouring CRISPR–Cas systems, many people have immune systems that are already primed to attack and disable the Cas9 protein. That could pose a challenge. According to Sontheimer, anti-CRISPR proteins could be prone to the same rejection issue, potentially imperilling the technology and triggering dangerous, inflammatory reactions in patients.
Other types of CRISPR inhibitor shouldn’t have the same limitation. And that diversity could be important in medical applications: for example, in limiting the editing activity of gene-targeted medicines, or fashioning phage therapies capable of wiping out difficult-to-treat bacteria without being stymied by the pathogen’s own CRISPR defences. It might also help in other proposed applications of CRISPR-blocking technologies.
But concerns over unforeseen ecological impacts abound. Many public officials and researchers also worry about gene drives being weaponized to wipe out agricultural systems or to spread a deadly disease. Anti-CRISPRs could provide a molecular safety net against these potential bio-attacks, says Sandia biochemist Joe Schoeniger. “You need to have an off-button,” he says. For now, such applications are mostly hypothetical. The only published report of researchers using anti-CRISPR proteins to inhibit a gene drive comes from a proof-of-principle experiment in yeast.
However, the idea is gaining traction. Hence, DARPA, in 2017, launched the Safe Genes program, a four-year, US$65-million initiative aimed at combating the dangers of CRISPR technologies. This has involved discovering new inhibitors against all types of CRISPR–Cas system and finding anti-CRISPRs that function in unique and useful ways.
As bioengineers continue to tinker with anti-CRISPRs, and as companies such as Acrigen move to introduce the inhibitors into therapeutic platforms, some biologists have also begun to grapple with more philosophical questions about the evolution of CRISPR–Cas systems in the first place. If bacteria with intact CRISPR protections commonly harbour phage-derived sequences for inhibitors that neutralize this immunity, then “CRISPR is clearly not doing its defence role in many of those cases”, says Edze Westra, who studies the ecology of CRISPR systems at the University of Exeter, UK. And yet, natural selection seems to maintain the system in working order. So, he asks, “what is its role apart from fuelling biotech start-up companies?”
These bedevilling mysteries won’t halt the steady march of CRISPR gene editing into human therapeutics, pest control and more. And for many, that’s why anti-CRISPRs are so important.
“There needs to be this shift to really controlling these editors so we make sure that you get the change you want and nothing else,” says Doudna. And just as the CRISPR–Cas systems that ushered in a biotechnology revolution started with a few curious observations in a laboratory, she notes, so too did the discovery of the inhibitors that could be a much-needed corrective.
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