How the interaction after PAM guides Cas9 binding and release from DNA

Two stable interactions of Cas9 with DNA were identified, including one located 14 base pairs downstream of the PAM.

• Cas9 interacts with DNA through specific binding sites crucial for its activity.
• One critical interaction site is positioned downstream of the protospacer adjacent motif (PAM), affecting Cas9's binding.
• Disruption of the post-PAM interaction site impairs Cas9's ability to bind and cleave DNA.
• A downstream helicase can displace DNA-bound Cas9 by targeting the weak post-PAM interaction.
• These findings may inform new strategies for modulating Cas9 activity.

Simplified