Enzymatic assembly for CRISPR split-Cas9 system: The emergence of a Sortase-based split-Cas9 technology

Area of Science:

• Molecular Biology
• Biotechnology
• Gene Editing

Background:

• CRISPR-Cas9 technology faces challenges with large Cas9 size and precise cellular control.
• Split-Cas9 systems improve enzyme delivery but struggle with controlled reassembly and activity.
• Existing methods often require chemical or physical induction for Cas9 activity.

Purpose of the Study:

• To develop an enzymatic split-Cas9 system using Sortase for controlled protein reconstitution.
• To enable precise genome editing in specific cell types without chemical induction.
• To explore alternative delivery methods and protein modification applications.

Main Methods:

• Developed an enzymatic split-Cas9 system utilizing the Sortase enzyme.
• Validated the system in E. coli, HEK cells, and Jurkat cells.
• Investigated nuclear localization signals, inducible promoters, mRNA/protein delivery, and S/MAR minicircle technology.

Main Results:

• Demonstrated successful assembly and activity of the Sortase-ligated Cas9 enzyme.
• Achieved precise genome editing in various cell types.
• Showcased the potential for Sortase in posttranslational modifications and protein assembly.

Conclusions:

• The Sortase-based split-Cas9 system offers enhanced control and efficiency for genome editing.
• This enzymatic approach provides a versatile platform for targeted gene editing and protein engineering.
• Sortase enzyme shows promise for applications beyond gene editing, including protein assembly in human cells.