So far, clinical applications of CRISPR-based gene editing have been largely limited to editing a person's cells outside of their body and then returning them. One reason for that limitation is that the safest, most accurate gene-editing tools don't fit inside viral delivery mechanisms that could target them to specific cell types or tissues inside the human body.
A University of Texas at Austin research team funded by the National Institutes of Health (NIH) and in a partnership with Bay Area biotech company Metagenomi Thereaputics has developed a smaller pair of "molecular scissors" for gene editing that could make site-specific delivery within the body possible. They described their results in the journal Nature Structural & Molecular Biology .
Metagenomi researchers identified a naturally occurring bacterial nuclease enzyme, Al3Cas12f, with particularly effective gene-editing capabilities for its size and small enough to fit into viral delivery systems. After performing an in-depth analysis, UT researchers modified the nuclease, producing an engineered variant with even better performance in human cells.
"We uncovered mechanistic features that explain why some Cas12f enzymes are more efficient than others," said David Taylor, a UT molecular biosciences professor and study co-author, referring to the group of CRISPR-based molecular scissors they studied. "With this understanding, we can begin to rationally design improved variants that outperform existing tools while maintaining a compact size that is ideal for delivery. Importantly, we also identified Al3Cas12f as a highly efficient nuclease across multiple genomic targets, making it a strong candidate for future therapeutic development.
Scientists can program adeno-associated virus (AAV) vectors to deliver instructions for building proteins, like CRISPR nucleases, to a specific target, be it a certain type of cell or tissue. However, there is a limit on the length of these instructions of about 1,000 protein building blocks, or amino acids, and the best nucleases currently don't make the cut.
Therefore, CRISPR's clinical applications to date are mostly for tissues that can be removed from and modified outside of the body such as blood and bone marrow.
Researchers have been searching for alternatives. A group of smaller nucleases called Cas12f, ranging from 400 to 700 amino acids long, has shown promise, but they have not proved themselves to be viable gene editors in the chaotic environment of real human cells, until now.
In the new study, the authors found that Al3Cas12f, coming from a specific type of bacteria, is capable of highly efficient editing surpassing two other Cas12f enzymes that researchers recently deployed via AAV to modify muscular dystrophy-associated genes in mice.
To find out what made this nuclease different from the rest, the team applied an imaging technique called cryo-electron microscopy and machine learning tools to build models that simulated the nuclease's structure and function as it forms and then interacts with DNA.
The authors learned that, compared to the two other enzymes, Al3Cas12f exhibited an extra-large interface between all of its components, allowing for a more secure connection.
"The expanded interface means the enzyme is much more stable. Compared to the others we looked at, Al3Cas12f basically comes preassembled and ready to go shortly after its pieces are produced," Taylor said.
While a sturdier molecular machine, the nuclease still struggled to edit some genes that the researchers targeted. Having analyzed the nuclease inside and out, the authors began to tinker with its makeup. Of the many variants they produced, one known as Al3Cas12f RKK stood above the rest.
The team introduced instructions for RKK directly into a line of human cells originally isolated from a patient with leukemia. Mutations in several of the genes they aimed to edit were associated with diseases such as cancer, atherosclerosis and amyotrophic lateral sclerosis (ALS).
The researchers saw that, across the tested targets, RKK improved on the original's editing efficiency of less than 10% to more than 80%.
The authors expect to build on their encouraging results. They next plan to conduct tests of the nuclease's performance when packaged into AAV vectors, which, if successful, could bring gene-editing therapy for many diseases much closer to reality.
Other UT authors are Kaoling Guan, Rodrigo Fregoso Ocampo, Tyler Dangerfield, Matthew Hooper, Madeline West, Nathan Appleby, Isabella Krudop and Kenneth A. Johnson.
This research was supported in part by the NIH's National Institute of General Medical Sciences (NIGMS).
"Smart delivery of gene-editing systems is a powerful notion with broad clinical implications, and this basic science finding takes us a significant step toward that future," said Erica Brown, Ph.D., acting director of NIGMS.
Adapted from a press release by the National Institutes of Health.
