Programmed genome editing by a miniature CRISPR-Cas12f nuclease

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• RNA-guided CRISPR-associated (Cas) nucleases are powerful genome editing tools.
• Large Cas9 and Cas12a nucleases face limitations in adeno-associated virus (AAV) delivery due to cargo size constraints.
• Development of smaller CRISPR systems is crucial for expanding therapeutic applications.

Purpose of the Study:

• To identify and characterize a novel, miniature CRISPR-Cas genome editing system.
• To evaluate the efficacy of this new system in bacterial and human cells.
• To assess its potential for therapeutic genome editing applications.

Main Methods:

• Identification of a class 2 type V-F CRISPR-Cas system from Acidibacillus sulfuroxidans (AsCas12f1).
• Characterization of AsCas12f1's RNA-guided endonuclease activity and protospacer adjacent motif (PAM) recognition (5' T-rich).
• Testing AsCas12f1 genome editing efficiency in bacteria and human cells using plasmid, ribonucleoprotein (RNP), and AAV delivery.

Main Results:

• AsCas12f1, a 422-amino acid nuclease, was identified and characterized.
• AsCas12f1 successfully performed RNA-guided DNA cleavage with staggered double-stranded breaks.
• Effective genome editing was demonstrated in both bacterial and human cells via multiple delivery methods, including AAV.

Conclusions:

• The miniature AsCas12f1 system provides a compact and versatile alternative to larger CRISPR nucleases.
• Its small size enhances suitability for AAV-mediated gene therapy and other delivery-constrained applications.
• AsCas12f1 characterization paves the way for engineering more compact and efficient genome manipulation technologies.