For all its supposed genetic editing finesse,
CRISPR’s a brute. The Swiss Army knife of gene editing tools chops up
DNA strands to insert genetic changes. What’s called “editing” is
actually genetic vandalism—pick a malfunctioning gene, chop it up, and wait for the cell to patch and repair the rest.
It’s a hasty, clunky process, prone to errors and other unintended
and unpredictable effects. Back in 2019, researchers led by Dr. David
Liu at Harvard decided to rework CRISPR from a butcher to a surgeon, one that lives up to its search-and-replace potential. The result is prime editing, an alternative version of CRISPR with the ability to “make virtually any targeted change in the genome of any living cell or organism.”
It’s the nip-tuck of DNA editing: with just a small snip on one DNA
chain, we have a whole menu of potential genetic changes at our
Prime editing was hailed as a fantastic “yay, science!” moment that could conceivably repair nearly 90 percent
of over 75,000 diseases caused by genetic mutations. But even at its
birth, Liu warned that CRISPR prime was only taking its first toddler
steps into the big, wild world of changing a life form’s base code.
“This first study is just the beginning—rather than the end—of a
long-standing aspiration in the life sciences to be able to make any DNA
change at any position in an organism,” he told Nature at the time.
Flash forward two years. Liu’s gene editing ingénue took some
stumbles. Despite its precise and effective nature, prime editing could
only edit genes in certain types of cells, while being less effective
and introducing errors in others. It also failed when trying to make
large genetic edits, particularly those that require hundreds of DNA
letters to be replaced to fix a disease-causing genetic mistake.
But the good news? Toddlers grow up. This week, three separate
studies advanced prime editing, helping the CRISPR tool grow into a more
sophisticated DNA-editing genius.
Two teams, based at the University of Massachusetts Medical School and the University of Washington,
reworked the tool’s molecular makeup to precisely cut out up to 10,000
DNA letters in one go—a challenge for prime editing 1.0. A third study from the tool’s original inventor probed its inner molecular workings,
identifying protein friends and foes inside the cell that control the
tool’s genetic editing abilities. By promoting friendly interactions,
the team increased prime editing’s efficiency in seven different cell
types nearly eight-fold. Even better, the “foes” that block prime’s
editing potential were identified using CRISPR—in other words, we’re
witnessing a full circle of innovation whereby gene editing tools help
build better gene editing tools.
Prime editing burst onto the gene editing scene for its dexterity and
precision. If the original CRISPR-Cas9 is a dancer with two left feet,
prime editing is a highly-trained ballerina.
The two processes start similarly. Both rely on a molecular “zip
code” to target the tool to a specific gene. In CRISPR, it’s called a
guide RNA. For prime editing, it’s a slightly modified version dubbed
Once the guides tether their respective dance partners to the gene,
their routines differ. For CRISPR, the second component, Cas9, acts as a
pair of scissors to snip both DNA strands. From here, cells can either
throw out parts of a gene, or—when given a template—insert a healthy
version of a gene to replace the original one. The cost is molecular
surgery. Just as an incision might not fully heal, a double-stranded
break to the DNA can introduce errors into the genetic code, leading to
unexpected effects that vary between cells.
Prime editing was the sophisticated upgrade set to fix that. Rather
than cutting both DNA strands, it lightly nips one chain. From there, it
can delete or insert genetic code based on a template without relying
on the cell’s DNA repair mechanism. In other words, prime editing opened
a new universe of genetic edits that’s far more controllable than
previous iterations. The original CRISPR typed with its eyes closed,
allowing small insertions, deletions, or mis-typed DNA letters into our
genetic text. With prime, we had found a true “search-and-replace”
editing tool—or so we hoped.
After releasing prime editing into the wild, scientists soon realized
that the wunderkind had its quirks. Although CRISPR prime made it
possible to precisely operate on DNA and change its text—replacing
letters, words, or even sentences—the tool struggled when editing larger
genes. In one estimate, larger genes comprise roughly 14 percent of
mutation types that are tough to treat with conventional CRISPR.
The two new studies tackled this conundrum. Published in Nature Biotechnology,
one team developed a method to precisely remove a large chunk of DNA by
adding an additional guide and restoring “scissor” activity to the Cas9
component. Here, the tool targeted two genetic spots at once to snip
out offending DNA. Similar to prime editing, the system, dubbed PEDAR,
was able build little DNA stretches through a special protein encoded in
it—like adding Lego blocks to snipped ends. Once the letter “blocks”
were long enough, they snapped into each other, bridging the gap after
snipping a gene.
“The advantage is that we can really expand the scale of editing—let
the ends of the genome find each other across a kilobase or 10
kilobases,” said Dr. Wen Xue, who led one of the studies.
Putting his money where his mouth was, Xue, with his team, next tried
PEDAR on a mouse model with inherited liver failure. In just a few
weeks, the mice began making a previously deficient protein to help
their liver cells grow into large chunks of healthy tissue. Treated with
PEDAR, the mice had minimal liver damage and kept a healthy weight
compared to siblings without genetic edits.
“That makes us very, very excited about this new technology,” said
Xue. “Before we were doing small fixes to treat small mutations. Here we
removed more than 1,300 base pairs, so that’s a very large genome
In another study, Dr. Junhong Choi reworked prime editing into
PRIME-Del. This version uses two RNA guides that grab onto the start and
end of a DNA chunk that needs to be cut out. Imagine a piece of rope
that’s too long and sagging in the middle. You snip out those parts and
paste the rest back together by adding glue to the end “flaps.” This
team’s prime editing upgrade was able to knock out up to 10,000 DNA
letters in human kidney cells.
Finally, the original inventor of prime editing also pitched in. One
problem, he said, is that the tool doesn’t work effectively across
multiple cell types and edits. It’s like a Word-editing keyword that
sometimes sticks. In this study,
his team tapped CRISPR as a way to figure out what’s been limiting the
original prime. With a method dubbed “CRISPR interference,” they
simultaneously disrupted hundreds of genes in cells to see how changing
the cell’s inner workings altered the gene editing tool’s capability.
With the test, the team teased out several proteins that block prime
editing, but also found a few that boost the tool’s effectiveness and
precision. Incorporating those updates into a next generation, the team
engineered PEmax (for “precision editing max”). In nearly 200 diverse
gene editing tasks, the upgraded prime editing tool enhanced efficiency
nearly 10-fold compared to the original.
Liu’s excited to see the tool evolving. To STAT News, he said the upgrades are “elegant variations of prime editing” that could
correct the vast majority of known disease-causing genetic insertions.
But perhaps the most mind-blowing part of this all? Similar to AI,
our current CRISPR tools are impacting the development of newer and more
powerful genetic editors in a virtuous circle. Let’s see where it
Image Credit: NIH Image Gallery