A National Institutes of Health (NIH)-funded research team has discovered an enhanced CRISPR gene-editing system that could enable targeted delivery inside the human body - a key step toward broader clinical use. Researchers identified a naturally occurring enzyme, Al3Cas12f, that is small enough to fit into adeno-associated virus vectors, a leading targeted delivery method for gene therapies. They then engineered an enhanced version that dramatically improved gene-editing performance in human cells.
The advance addresses a major limitation in CRISPR technology. Commonly used gene-editing proteins are too large for targeted delivery systems, restricting clinical applications to cells modified outside the body, such as blood and bone marrow.
"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 NIH's National Institute of General Medical Sciences (NIGMS).
Using imaging and machine learning tools, researchers at the University of Texas at Austin analyzed the enzyme's structure. They found it forms a more stable and tightly connected complex than other enzymes of a similar size, allowing it to function more effectively in human cells.
"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," said corresponding author David Taylor, Ph.D., a molecular bioscience professor at UT Austin.
The team then engineered a variant, known as Al3Cas12f RKK, which significantly improved editing efficiency from less than 10% to more than 80% across tested targets. In a commonly edited region of the genome, efficiency reached 90%.
Of the many variants the team produced, 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 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.
This research was supported in part by NIGMS through grant R35GM138348.
