Repurposing CRISPR-Cas12b for mammalian genome engineering.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas systems are prokaryotic adaptive immune mechanisms utilized for genome engineering.
• CRISPR-Cas12b (C2c1) is a Type V-B system active in vitro, but its endogenous genome cleavage and mammalian applicability were unexplored.
• Existing Cas12b orthologs have optimal temperatures above 40°C, limiting their use in mammalian systems.

Purpose of the Study:

• To identify and characterize a novel CRISPR-Cas12b system suitable for mammalian genome engineering.
• To evaluate the temperature range and cleavage activity of the identified Cas12b system.
• To assess the specificity and efficiency of the Cas12b system in mammalian genome editing applications.

Main Methods:

• Identification and cloning of a Cas12b system from Alicyclobacillus acidiphilus (AaCas12b).
• In vitro characterization of AaCas12b nuclease activity across a temperature range.
• Application of AaCas12b for mammalian genome editing, including single/multiplex editing, gene activation, and generation of mutant mouse models.
• Whole-genome sequencing to assess AaCas12b specificity and off-target effects.

Main Results:

• The identified AaCas12b exhibits optimal nuclease activity between 31°C and 59°C, suitable for mammalian applications.
• AaCas12b demonstrated versatility in mammalian genome engineering, enabling single and multiplex editing, gene activation, and creation of gene mutant mouse models.
• Whole-genome sequencing confirmed high specificity and minimal off-target mutations associated with AaCas12b-mediated editing.

Conclusions:

• The AaCas12b system is a novel and versatile CRISPR tool for mammalian genome engineering.
• Its broad optimal temperature range and high specificity make it advantageous over previously reported Cas12b systems.
• AaCas12b expands the CRISPR toolbox for advanced genetic research and therapeutic applications in mammals.