Miniaturized CRISPR Packs a Mighty Gene Editing Punch

When the gene editing tool CRISPR-Cas9 rocketed to fame more than a decade ago, it transformed biotechnology. Faster, cheaper, and safer than previous methods, the tool helped scientists gain insight into gene functions—and when they go wrong.

CRISPR also brought the potential to change the lives of people living with inherited diseases. Thanks to its gene editing prowess, the tool can supercharge immune cells’ ability to hunt down cancer and other rogue cells. In late 2023, the FDA approved a CRISPR therapy for sickle cell disease and later gave the greenlight to people with a blood disorder called transfusion-dependent beta thalassemia. Many more therapies are in the works.

But CRISPR has a hefty problem: The system is too large, making it difficult to deliver the gene editor to cells in muscle, brain, heart, and other tissues.

Now, a team at Mammoth Biosciences has a potential solution. Cofounded by CRISPR pioneer Jennifer Doudna at the University of California, Berkeley, the company has long sought to downsize the original CRISPR-Cas9 system. Their new iteration, dubbed NanoCas, slashed the size of one key component, Cas9, to roughly one-third of the original.

The slimmed-down setup allowed the tool to be packaged into a single “delivery box”—a virus that’s commonly used for gene therapy inside the body. In mice and monkeys, the team used NanoCas to edit genes involved in inherited high cholesterol and Duchenne muscular dystrophy.

“CRISPR gene editing is a transformative technology for addressing genetic diseases, but delivery constraints have largely limited its therapeutic applications to liver-targeted and ex vivo [outside the body] therapies,” wrote the team in a preprint describing their results. The compact NanoCas “opens the door” for editing tissues inside the body.

Delivery Woes

CRISPR has two main components. One is an RNA molecule that’s like a bloodhound, seeking out and tethering the setup to a target DNA section. Once docked, the second component, a Cas protein, slices or snips the genetic ribbon.

Over the years, scientists have discovered or engineered other versions of Cas proteins. Some target RNA, the “messenger” that translates genes into proteins. Others swap out single genetic letters causing inherited diseases. Some even recruit enzymes to modify the epigenome—the system controlling which genes are turned on or off.

All these tools have a major problem: They’re difficult to deliver inside the body because of their size. Current CRISPR therapies mainly rely on extracting cells and swapping their genes inside petri dishes. The edited cells are infused back into the patient. Called “ex vivo” therapy, these treatments mainly focus on blood-based disorders.

Correcting genetic problems inside the body with CRISPR adds to the complexity. Most therapies focus on the eyes or the liver, which are both relatively easy to access with a shot. For all other tissues, delivery is the main problem.

To shuttle the editors to tissues and cells, they have to be packaged inside a virus or a fatty bubble. Cas proteins can reach over a thousand amino acids in length, which already stresses the capacity of the delivery vehicles. Add in guide RNA components, and the system exceeds luggage limits.

To get around weight restrictions, scientists have encoded the guide RNA and Cas components separately into two viral carriers, so both can sneak into cells. Alternatively, they’ve used fatty bubbles called liposomes that encapsulate both gene editing components.

Neither is perfect. A double load of virus increases the risk of an immune response. Liposomes generally end up in the liver and release their cargo there. This makes them excellent at editing genes in the liver—for example, PCSK9, to treat high levels of cholesterol—but they struggle to reach other tissues. Important targets such as the brain and muscles are out of reach.

Small But Mighty

Why not shrink the cargo so it fits into the same viral luggage?

Here, Mammoth Biosciences searched metagenomics databases for smaller Cas proteins. These databases contain diverse samples from across the planet, including from microbes gathered in swamps, seawater, our guts, and other sources. The team looked for systems that could edit as efficiently as Cas9, required only a tiny guide RNA component, and were under 600 amino acids.

From 22,000 metagenomes, the team zeroed in on 176 candidates. Each was vetted in human kidney cells in a dish—rather than using bacteria, which is the norm. This screens for Cas variants that work well inside mammalian cells, which is a common bottleneck, wrote the team.

After more tests, they landed on NanoCas. It worked with roughly 60 percent of the RNA guides they tried out, and after some tinkering, easily sliced up targeted DNA.

The tiny editor and its guide RNA fit into a single viral vector. As proof of concept, the team made a NanoCas system targeting PCSK9, a gene associated with dangerously high levels of cholesterol, in the livers of mice. Delivered in a single injection into the veins, the tiny tool slashed the gene to undetectable levels in the blood.

Next, the team turned to a gene called dystrophin in muscles, a tissue traditional CRISPR methods struggle to reach. In Duchenne muscular dystrophy, mutated dystrophin causes progressive muscle loss. NanoCas edited the gene across a wide variety of muscle types—thigh, heart, and calf muscle. The efficacy varied, ranging from 10 to 40 percent of edited cells.  

The team next tested NanoCas in monkeys. After about two months, roughly 30 percent of their skeletal muscle cells were edited. Heart cells were less responsive, with only half the efficacy.

“To our knowledge,” this is the first time someone has edited muscles in a non-human primate with a single virus CRISPR system, wrote the team.

Gene therapies using delivery viruses can tax the liver, but throughout the trial the monkey’s liver functions and other health factors stayed relatively normal. But many questions remain. Although the system edited targeted genes in healthy monkeys, whether it can treat genetic muscle loss remains to be seen. As with other gene editing systems, there’s also the risk of unintentionally editing non-targeted genes or spurring an immune attack.

That said, the miniature NanoCas—and potentially other tiny Cas proteins yet to be discovered—could shuttle CRISPR to a variety of tissues in the body with a jab. The team is already exploring the system’s potential for targeting brain diseases. The technology could also be reworked for use in epigenetic or base editing.

Above all, the study suggests small Cas proteins can be mighty.

“NanoCas demonstrates that carefully selected compact systems can achieve robust editing across various contexts, challenging the assumption that small CRISPR systems are inherently less effective,” wrote the team.