Engineering the MmeFz2-ωRNA system for efficient genome editing through an integrated computational-experimental...

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Eukaryotic Fanzor proteins are RNA-guided nucleases with genome editing potential.
• Current Fanzor systems exhibit suboptimal efficiency in mammalian cells, limiting their therapeutic applications.

Purpose of the Study:

• To optimize the MmeFz2-ωRNA Fanzor system for enhanced genome editing in mammalian cells.
• To develop improved Fanzor variants for potential therapeutic applications.

Main Methods:

• Utilized AlphaFold3 for rational redesign of the ωRNA scaffold, resulting in a smaller and efficient structure.
• Employed structure-guided and AI-augmented protein engineering to generate novel Fanzor variants (enMmeFz2, evoMmeFz2).
• Fused Fanzor variants with the HMG-D DNA-binding domain to further enhance editing performance.

Main Results:

• Minimized ωRNA scaffold maintained high efficiency (up to 82.2%) and was 30% smaller.
• Engineered Fanzor variants (enMmeFz2, evoMmeFz2) showed an average 32-fold increase in activity across 38 genomic loci.
• Fanzor-HMG-D fusions demonstrated enhanced editing, with evoMmeFz2-HMG-D successfully restoring dystrophin in a Duchenne muscular dystrophy mouse model via AAV delivery.

Conclusions:

• Fanzor2 represents a promising gene editing platform for genome engineering and therapeutic development.
• AI-guided engineering significantly accelerates the development of genome editors and reduces experimental workload.
• Optimized Fanzor systems hold potential for treating genetic disorders like Duchenne muscular dystrophy.