Structural visualization of the molecular evolution of CRISPR-Cas9

Area of Science:

• Molecular Biology
• Structural Biology
• Evolutionary Biology

Background:

• RNA-guided nucleases like Cas9 and IscB are key components of CRISPR-Cas and OMEGA systems, respectively.
• Previous studies suggested IscB evolved into Cas9 through protein expansion and guide RNA miniaturization.
• The precise evolutionary pathway remained unclear.

Purpose of the Study:

• To elucidate the evolutionary transition of RNA-guided nucleases from IscB to Cas9.
• To determine the structural basis for the functional divergence between IscB and Cas9.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine the structures of four diverse RNA-guided nucleases (two IscBs and two Cas9s).
• Structures were analyzed in complex with cognate guide RNA and target DNA.
• Comparative structural analysis was performed with existing IscB and Cas9 structures.

Main Results:

• The evolution from IscB to Cas9 involved loss of the PLMP domain and acquisition of the REC3, bridge helix extension, and REC1 domains.
• These changes expanded the REC lobe, enhancing target DNA cleavage specificity.
• Structural conservation of RNA scaffolds suggests CRISPR-Cas9's dual RNA guides evolved from OMEGA system's single ωRNA guides.

Conclusions:

• The study reveals a stepwise structural evolution from IscB to Cas9, explaining the development of adaptive immunity systems.
• Findings provide insights into the exaptation of transposon-associated nucleases for immune functions.
• The structural modifications underscore the increased specificity and complexity of Cas9 compared to IscB.