Rational design of a split-Cas9 enzyme complex.

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• The clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune system utilizes Cas9, an RNA-guided DNA endonuclease.
• Cas9 is a powerful tool for genome editing, transcriptional regulation, and cellular imaging, featuring a bilobed structure that changes conformation upon binding guide RNA and DNA.

Purpose of the Study:

• To investigate the molecular mechanisms and significance of Cas9's interlobe rearrangement in target recognition and DNA cleavage.
• To engineer a split-Cas9 system for enhanced control and regulatability in genome engineering applications.

Main Methods:

• Designed and expressed a split-Cas9 enzyme, separating the nuclease and alpha-helical lobes into distinct polypeptides.
• Investigated the ability of single-guide RNA (sgRNA) to recruit the separated lobes into a functional ternary complex.
• Utilized a modified sgRNA to disrupt split-Cas9 dimerization and activity, enabling the development of an inducible dimerization system.

Main Results:

• The separated Cas9 lobes did not interact independently but were effectively recruited by sgRNA into a functional complex.
• The reconstituted split-Cas9 enzyme successfully recapitulated the site-specific DNA cleavage activity of full-length Cas9.
• A modified sgRNA prevented split-Cas9 dimerization, demonstrating the potential for inducible control over nuclease activity.

Conclusions:

• The study successfully demonstrates the feasibility of a split-Cas9 system that can be reconstituted by sgRNA for precise DNA cleavage.
• Split-Cas9 offers a highly regulatable platform for advanced genome engineering, allowing for inducible control over nuclease activity.
• This approach provides a novel strategy for developing safer and more precise genome editing tools.