DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics
• Genome Engineering

Background:

• The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is a powerful RNA-guided endonuclease.
• Cas9-guide RNA complexes are widely used for genome engineering in diverse organisms.
• Understanding the precise mechanism of Cas9 DNA interrogation is essential for its efficient application.

Purpose of the Study:

• To elucidate the molecular mechanisms by which Cas9-RNA complexes interact with DNA.
• To determine how Cas9 identifies specific DNA cleavage sites.
• To investigate the role of the protospacer adjacent motif (PAM) in Cas9 binding and activity.

Main Methods:

• Single-molecule biochemical experiments.
• Bulk biochemical assays.
• Competition assays to study DNA binding and strand separation dynamics.

Main Results:

• Cas9-RNA binding and DNA cleavage are dependent on the recognition of a trinucleotide protospacer adjacent motif (PAM).
• DNA binding affinity correlates with PAM density; sequences lacking a PAM are not targeted, even if complementary to the guide RNA.
• PAM recognition initiates DNA strand separation and RNA-DNA heteroduplex formation, proceeding directionally, and triggers Cas9 catalytic activity.

Conclusions:

• Cas9 utilizes PAM recognition as a critical step for rapid scanning of large DNA molecules and identification of potential target sites.
• The PAM sequence acts as a regulatory element, controlling the initiation of DNA cleavage by Cas9.
• These findings provide fundamental insights into the specificity and regulation of Cas9-mediated genome editing.