Structural insights into Cas9-mediated prespacer selection in CRISPR-Cas adaptation

Area of Science:

• Microbiology
• Molecular Biology
• Structural Biology

Background:

• CRISPR-Cas systems provide adaptive immunity in prokaryotes.
• Spacer acquisition, a key adaptation step, involves integrating foreign DNA into the CRISPR array.
• Cas1-Cas2 integrase and accessory proteins mediate spacer acquisition, with Cas9 involvement noted in type II-A systems.

Purpose of the Study:

• To elucidate the structural mechanism of Cas9-mediated prespacer selection in Streptococcus thermophilus type II-A CRISPR-Cas systems.
• To characterize the composition and conformation of the prespacer selection supercomplex.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was employed to determine high-resolution structures.
• Structural analysis focused on the Streptococcus thermophilus type II-A prespacer selection supercomplex.

Main Results:

• Structures of the supercomplex in DNA-scanning and two distinct protospacer adjacent motif (PAM)-bound states were resolved.
• The findings provide atomic-level insights into how Cas9 directs the selection of specific DNA sequences during adaptation.
• The study highlights the formation of a Cas9-Cas1-Cas2-Csn2 supercomplex.

Conclusions:

• Cas9 plays a crucial role in prespacer selection by forming a supercomplex with integrase and accessory proteins.
• The structural data reveals the mechanism of Cas9-mediated DNA target selection during CRISPR-Cas adaptation.
• This work demonstrates the plasticity of Cas9 and expands understanding of its biological functions beyond gene editing.