DNA targeting by compact Cas9d and its resurrected ancestor

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• Type II CRISPR endonucleases are essential programmable genome editing tools.
• Discovery of compact nucleases like Cas9d (a type II-D nuclease) expands CRISPR system diversity.

Purpose of the Study:

• To determine the cryo-electron microscopy (cryo-EM) structures of Cas9d nuclease in various functional states.
• To elucidate the stepwise DNA targeting mechanism and the role of guide RNA.
• To engineer novel compact nucleases for enhanced genome editing.

Main Methods:

• Cryo-electron microscopy (cryo-EM) for structural determination.
• Ancestral sequence reconstruction for protein engineering.
• In vivo genome editing assays in mammalian cells.

Main Results:

• Detailed cryo-EM structures of Cas9d (747 amino acids) reveal a stepwise DNA targeting process involving a conformational switch.
• The guide RNA acts as a structural scaffold, and its length can be reduced by ~25% while maintaining in vivo activity.
• Engineered compact nucleases derived from ancestral sequence reconstruction demonstrate efficient genome editing in mammalian cells.

Conclusions:

• The study provides mechanistic insights into the DNA targeting of diverse type II CRISPR-Cas systems.
• The findings offer a blueprint for re-engineering minimal RNA-guided DNA endonucleases for genome editing applications.