Engineered Un1Cas12f1 for multiplex genome editing with enhanced activity and targeting scope.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• The CRISPR-Cas12f system shows promise for AAV-delivered gene therapy.
• Its use is limited by narrow PAM recognition (TTTR) and lower editing efficiency.

Purpose of the Study:

• To engineer an optimized CRISPR-Cas12f variant with expanded PAM recognition and improved editing efficiency.
• To demonstrate the utility of the engineered variant for various genome engineering applications.

Main Methods:

• Bacterial library screening was employed to identify beneficial mutations.
• Mammalian cell validation confirmed the performance of the engineered variant.
• The system was adapted for transcriptional activation and base editing.

Main Results:

• The engineered evoCas12f variant exhibits expanded PAM recognition to NTNR/NYTR.
• It shows 1.4-fold enhanced activity at TTTR sites and up to 91% editing efficiency.
• Homozygous mutations were efficiently generated in F0 mice, even at non-canonical PAM sites.

Conclusions:

• evoCas12f is a compact, highly efficient CRISPR platform.
• It enables multiplexed editing and high-resolution targeting.
• This advancement expands possibilities for therapeutic genome engineering.