Engineered Un1Cas12f1 with boosted gene-editing activity and expanded genomic coverage.

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• Compact programmable nucleases are valuable for genome editing and hold therapeutic promise, especially when delivered via adeno-associated virus (AAV) vectors.
• Current nucleases face limitations due to suboptimal editing efficiency and strict protospacer-adjacent motif (PAM) requirements, hindering practical applications.
• The development of more efficient and versatile genome editing tools is crucial for advancing gene therapy.

Purpose of the Study:

• To engineer an optimized variant of Un1Cas12f1 with improved editing activity, reduced off-target effects, and expanded PAM specificity.
• To evaluate the efficacy of the engineered nuclease, MiniCasUltra, for simultaneous in vivo gene editing in a mouse model.
• To assess the therapeutic potential of MiniCasUltra for treating neovascular age-related macular degeneration.

Main Methods:

• Rational mutagenesis was employed to engineer MiniCasUltra from Un1Cas12f1.
• The editing activity and off-target effects of MiniCasUltra were compared to existing tools.
• Simultaneous gene editing of *Pten* and *Fah* in mouse liver was achieved using a single AAV vector encoding MiniCasUltra and two single-guide RNAs.
• MiniCasUltra was delivered via AAV to target vascular endothelial growth factor A in a mouse model of choroidal neovascularization.

Main Results:

• MiniCasUltra demonstrated a sixfold increase in editing activity compared to Un1Cas12f1.
• The engineered nuclease exhibited minimal off-target effects with an on/off-target ratio greater than 10.
• MiniCasUltra possesses an expanded PAM preference of 5'-WBTR, including noncanonical sites like 5'-TCTG.
• Simultaneous editing of *Pten* and *Fah* in mouse liver yielded significantly higher indel rates (15.82% and 29.39%) than CasMINI V3.1.
• AAV delivery of MiniCasUltra successfully reduced choroidal neovascularization lesions in a mouse model.

Conclusions:

• MiniCasUltra represents a significant advancement in genome editing technology, offering enhanced efficacy and versatility.
• Its compact size, high activity, and broad targeting range make it suitable for in vivo applications.
• MiniCasUltra shows considerable potential for developing novel therapeutic interventions for genetic diseases and conditions like age-related macular degeneration.