STRUCTURAL BIOLOGY. A Cas9-guide RNA complex preorganized for target DNA recognition

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• Bacterial adaptive immunity relies on CRISPR-Cas systems to degrade foreign DNA.
• Type II CRISPR-Cas systems, including Cas9 endonuclease, are activated for DNA recognition via guide RNA binding through an unclear mechanism.

Purpose of the Study:

• To elucidate the structural mechanism of Cas9 activation upon guide RNA binding.
• To understand how the guide RNA's seed sequence influences Cas9 conformation and DNA engagement.

Main Methods:

• Determined crystal structures of Cas9 bound to single-guide RNA.
• Analyzed the conformation of Cas9 in apo, guide RNA-bound, and DNA-bound states.

Main Results:

• Cas9 bound to single-guide RNA adopts a unique conformation distinct from apo and DNA-bound states.
• The 10-nucleotide guide RNA seed sequence adopts an A-form conformation, pre-ordered for DNA interrogation.
• This pre-ordered seed sequence is critical for forming a DNA recognition-competent Cas9 structure.

Conclusions:

• The guide RNA seed sequence plays a pivotal role in priming Cas9 for target DNA recognition.
• This 'seed' mechanism in bacterial CRISPR-Cas9 shows convergent evolution with eukaryotic Argonaute proteins in RNA interference.