Visualizing how CRISPR-Cas9 has changed at the molecular level over time

Cryo-electron microscopy reveals structural changes involved in the evolution of IscB into Cas9.

• IscB, approximately 500 residues, evolved into Cas9, ranging from 700 to 1,600 residues, through protein expansion and guide RNA miniaturization.
• Evolution from IscB to Cas9 involved the loss of the N-terminal PLMP domain and the gain of the zinc-finger-containing REC3 domain.
• Subsequent structural alterations included bridge helix extension and REC1 domain acquisition, enhancing target DNA cleavage specificity.
• The conservation of RNA scaffolds suggests that the dual CRISPR RNA guides of Cas9 evolved from the single ωRNA guides of OMEGA systems.
• These findings highlight the sequence of structural changes that allowed transposon-associated RNA-guided nucleases to adapt for use in immune systems.

Simplified