Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

Area of Science:

• Microbiology
• Molecular Biology
• Immunology

Background:

• Prokaryotes possess adaptive immunity against viruses through CRISPR-Cas systems.
• CRISPR-Cas systems use small RNA molecules to guide Cas nucleases to target viral DNA.
• Streptococcus pyogenes Cas9 requires a protospacer adjacent motif (PAM) for DNA cleavage.

Purpose of the Study:

• To investigate the mechanism of spacer acquisition in CRISPR-Cas systems.
• To determine if Cas9 plays a role in selecting spacer sequences during acquisition.
• To elucidate the role of PAM recognition in the generation of prokaryotic immunological memory.

Main Methods:

• Genetic manipulation of Cas9 alleles to alter PAM recognition.
• Analysis of spacer acquisition specificity in Streptococcus pyogenes.
• Co-immunoprecipitation assays to study protein interactions within the acquisition machinery.

Main Results:

• Cas9 directly recognizes PAM sequences during spacer acquisition.
• Altering or removing the PAM recognition motif of Cas9 modified spacer acquisition specificity.
• Cas9 interacts with other acquisition proteins (Cas1, Cas2, Csn2) to confer PAM specificity.

Conclusions:

• Cas9 has a novel function in recognizing PAM sequences during the acquisition of new spacers.
• This PAM recognition by Cas9 is essential for the specificity of prokaryotic adaptive immunity.
• The study establishes a new mechanism for the generation of immunological memory in prokaryotes.