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Compact Gene Editing Enzyme May Improve Success Rates of Gene Therapies

NEW YORK – Japanese researchers have engineered new variants of the gene-editing enzyme Cas12f that are as effective as the molecular scissors Cas9 but are only one-third of its size.

The compact size of the new enzyme variants means that a greater quantity of the gene editing protein can be packaged into vectors such as adeno-associated viruses (AAVs) commonly used to infect living human cells requiring DNA editing.

As reported in Cell on Friday, investigators from the University of Tokyo, Kyoto Prefectural University of Medicine, Jichi Medical University, and elsewhere used deep mutational scanning (DMS) and structural analysis to create the small and efficient Cas12f variants.

The main reason for developing small-sized enzymes was due to the size limitations of AAVs and the limited gene editing cargo that can fit into them. "Cas9 is at the very limit of this size restriction, so there has been a demand for a smaller Cas protein that can be efficiently packaged into AAV and serve as a genome-editing tool," corresponding author Osamu Nureki from the Department of Biological Sciences at the University of Tokyo, said in a statement.

For their study, the researchers used DMS to identify over 200 mutations in amino acids that could enhance the activity of Cas12f. Next, using structure prediction models such as AlphaFold, they shortlisted two 'enhanced' variants, which they refer to as enAsCas12f.

These new variants have more than 10 times the genome-editing activity compared to the original Cas12f type and are comparable to Cas9, the authors added.

Meanwhile, their analysis of the cryoelectronic microscopy structures of the enhanced enzymes revealed that their mutations stabilized dimer formation and enabled robust interaction with nucleic acids, enhancing their ability to make snips in DNA.

Subsequent experiments in mice using the new enzymes, packaged with partner genes in an AAV, confirmed efficient knock-in and knockout effects. They also used the enhanced gene editors on human cell lines, which demonstrated their potential for in vivo gene therapies, especially for diseases such as hemophilia, they noted. 

Overall, the authors believe the DMS approach is more reliable and efficient for developing enzyme mutants with desired qualities than methods such as directed evolution with random mutagenesis. "By applying this powerful approach to other Cas enzymes with different PAM sequences, we can potentially generate efficient genome-editing enzymes capable of targeting a wide range of genes," they concluded.

Although the researchers discovered various potentially effective combinations for engineering an improved AsCas12f gene-editing system, they believe these may not have been the most optimal mixes. As a next step, they added that computational modeling or machine learning could be used to sift through the combinations and predict which might offer even better improvements.