What about more common diseases? Verve Therapeutics, a genome editing start-up, collaborated with Gaudelli’s team at Beam to test a one-shot strategy in two monkey models of heart disease. Verve used a lipid nanoparticle to deliver the ABE to the liver of crab-eating macaques to inactivate a pair of known genes that regulate cholesterol. CEO Sek Kathiresan reported a dramatic lowering of “bad” LDL cholesterol and triglycerides in animals targeted at the PCSK9 and ANGPTL3 genes, respectively.20 The results need to be confirmed and extended in humans, which is some years away. But base editing could help realize Kathiresan’s dream of a “one-and-done genome editing medicine for heart disease,” providing an alternative to chronic statins and reduce the 18 million cardiovascular deaths each year.
From Archimedes in the bath to Isaac Newton’s bruised head, there are many legendary aha moments in science history. Perhaps the most bizarre episode belongs to the late Kary Mullis, who recalled the invention of the polymerase chain reaction in a quite extraordinary Nobel lecture. It’s too good not to relive here:
One Friday night I was driving, as was my custom, from Berkeley up to Mendocino where I had a cabin far away from everything off in the woods… As I drove through the mountains that night, the stalks of the California buckeyes heavily in blossom leaned over into the road. The air was moist and cool and filled with their heady aroma… EUREKA!!!!… EUREKA again!!!!… I stopped the car at mile marker 46.7 on Highway 128. In the glove compartment I found some paper and a pen… “Dear Thor!” I exclaimed. I had solved the most annoying problems in DNA chemistry in a single lightning bolt… We got to my cabin and I started drawing little diagrams… with the aid of a last bottle of good Mendocino County cabernet, I settled into a perplexed semiconsciousness… The first successful experiment happened on December 16th. I remember the date. It was the birthday of Cynthia, my former wife… There is a general place in your brain, I think, reserved for “melancholy of relationships past.” It grows and prospers as life progresses, forcing you finally, against your grain, to listen to country music.21
By comparison, Andrew Anzalone’s story of the genesis of “prime editing”—fueled by caffeine not cabernet, ambling around the streets of Lower Manhattan rather than speeding through the Napa night—could use a little work in the dramatic license stakes. But his ideas, formed in 2017 before leaving Columbia University for a position in Liu’s lab, were crucial in devising a new genome editing platform. A physician-scientist rather than a chemist, Anzalone was inspired by Liu’s base editing exploits, but sensed an opportunity to go further. “The base editors were really good for making four possible base changes but they couldn’t address the other eight base changes or the small indels,” he said.22IV
In October 2019, Liu unveiled a new genome editing technology developed by Anzalone and other members of the lab that riffed on base editing, expanding the repertoire of potential DNA alterations. “This is the beginning of an aspiration to make any DNA change in any position of a living cell or organism,” Liu said.23 I was sitting in the audience of four hundred rapt scientists at the Cold Spring Harbor Laboratory for Liu’s first public presentation on prime editing, just ten days before the study was published in (no surprise) Nature.24 “Prime editing is somewhat complicated,” Liu admitted. But it works, and the meticulous four-step sequence offers important advantages.
There are more than 75,000 known disease-causing mutations in the human genome—about half of those are point mutations—but most can’t be targeted by CRISPR-Cas9 or base editing. While base editing’s strength is its ability to make a class of base substitutions known as transitions, they only account for four of the dozen possible base changes. The CBE would in principle fix 14 percent of known point mutations; the ABE accounted for a higher fraction, some 48 percent.
Anzalone wondered if he could build on this technology to engineer any single-base change, transitions and transversions.25 What he developed is a new system that moves scientists closer to a true search-and-replace function for DNA, regardless of the letter or its location. Naturally, the system would start with the programmable single-guide RNA, which directs Cas9 to the stretch of DNA to be edited. But what if, instead of providing the replacement sequence via a DNA template, he used the same guide RNA molecule to supply the edit? The system would have two programmable elements—the target site and the edit itself. Naturally the replacement sequence would have to be converted from the RNA guide to DNA, but luckily there’s a very well-known enzyme for that—reverse transcriptase (RT).V
The extended guide RNA, renamed the pegRNA (prime editing-gRNA), specifies the target as well as the desired edit. Similar to the construction of the base editors, Anzalone fused RT to dead Cas9, then figured out a scheme to coax the edited DNA copy into the target sequence. The first step is to engineer the pegRNA and RT enzyme to copy the pegRNA strand into DNA. This results in a flap of DNA that needs to be stitched into the double helix. (Helpfully, cells have enzymes called “flap nucleases” that help in this process.) Finally, the method introduces a nick into the complementary strand, which is then repaired to fully match the edited strand.
Anzalone’s approach didn’t get off to an auspicious start. When he fused RT to Cas9, he achieved zero editing. But further tests with a batch of RT variants soon resulted in some positive results. If the original CRISPR approach is a molecular scissors and base editing is a more precise pencil eraser, Liu describes prime editing as a word processor, capable of performing a search-and-replace function on any typo in the DNA alphabet. It also carries out some classes of insertions and deletions (indels), including those responsible for the most common form of cystic fibrosis (a three-base deletion) and Tay-Sachs disease (a four-base insertion), respectively.
After submitting their report to Nature, Liu and Anzalone had little trouble attending to the three anonymous referees’ comments. Figuring out the identity of one of them was easy: few reviewers sprinkle words like “quixotic” in their reports. Urnov effusively recommended publication, citing the increased flexibility on editing sites, no more PAM deserts, no DNA donor, few off-targets, and the early data on correcting DNA in neurons.
In their paper, the Liu group showcased the full range of prime editing’s prowess—175 different edits, including 100 point mutations of all possible types; repair of known disease mutations in human cells; insertions and deletions in the forty to eighty base range; and simultaneously deleting two bases while converting a G-to-a-T a few bases away. That’s like watching Lionel Messi slalom his way through the opposition defense and around the goalkeeper before tapping the ball into the net. Or as Sharon Begley put it, “the genome equivalent of a pool shark’s banking the 9 ball off the 7 and sinking the 1, 5, and 6.”26
Liu’s Cold Spring Harbor unveiling of prime editing was pretty much as close to a drop-the-mic moment as I’ve witnessed at a science meeting. He had given the meeting organizers a deliberately vague summary of his talk for the program book to preserve the element of surprise. I shook my head in astonishment as he flashed a pie chart showing the categories of human disease mutations, and said calmly that prime editing could in theory address 89 percent of them. Thirty minutes later, Liu wrapped up his talk by acknowledging Anzalone, with a smile: “I’m really looking forward to seeing what Andrew can do in the second year of his postdoc!” Doudna, who was sitting in the front row, later declared, “I literally had chills running down my spine” as she savored the latest power upgrade to the CRISPR toolbox.