EvoMax Improved Fanzor2, a Compact DNA Editor; Enhanced Variants Caused Toxicity in Mice
EvoMax Improved Fanzor2, a Compact DNA Editor; Enhanced Variants Caused Toxicity in Mice
On August 24, Nature Biotechnology published a paper on EvoMax, an iterative system in which a model selects protein variants for the next round of experiments. The authors worked with Fanzor2, a compact editing protein. A guide RNA directs it to a chosen DNA site, where it makes a cut. Its small size allows the editor and guide RNA to fit into a single AAV, a viral vector that carries genetic instructions.
Before this study, Fanzor2 showed low DNA editing activity in an unsorted cell population. A conventional search for effective mutations would have required large libraries of protein variants and high-throughput screening. For EvoMax, the researchers collected 209 activity measurements for single substitutions. The model combined these data with estimates of how well each substitution would be tolerated in related proteins and in the predicted Fanzor2 structure. In each of three rounds, the laboratory tested between 10 and 20 variants, and the best variant became the starting point for the next round.
The authors also shortened and stabilized the guide RNA and attached the human La protein to the editor to protect the RNA's exposed end. This produced the FanzMAX v3-hLa system. In a mixed population of human cells, it edited up to 97% of DNA at the best-performing site. The mean editing efficiency across 19 sites was about 33%.
The complete system was then tested in the livers of mice carrying the human version of the PCSK9 gene. The AAV 1.0 configuration produced about 25% editing in the liver and reduced blood PCSK9 protein levels by 34%, with no detected toxicity. Two more potent configurations caused acute toxicity, and the animals died within 7 to 14 days. After AAV 3.0 treatment, large deletions, defined as losses of DNA fragments longer than 30 base pairs, accounted for 36% of edited reads in the liver.
A report on Cas9d Ultra described another compact editor. Delivered in a single AAV9, it modified the Pcsk9 site in mouse liver at rates ranging from 8,3% to 28,9% and reduced LDL cholesterol. Cas9d Ultra replaces a single DNA base, whereas Fanzor2 cuts both strands. Similar editing rates achieved with a single vector therefore reflect different cellular DNA repair processes.
The AAV configurations differed simultaneously in the Fanzor2 variant, La protein, guide RNA, and vector elements. Selecting the next version requires considering the editing rate, the sequence changes that remain after the DNA cut is repaired, and the tissue response.