A PAM-Induced Signalling Activates the Communication between HNH and RUVC in CRISPR-Cas9

Abstract

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is a bacterial immune system, which has revolutionized life sciences through the introduction of a facile genome editing technology. In this system, the endonuclease Cas9 associates with guide RNAs to match and cleave complementary DNA sequences, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Thanks to a key DNA recognition element, a Protospacer Adjacent Motif (PAM), Cas9 performs site-specific DNA cleavages, enabling to engineer biological systems with unique efficiency and resulting in numerous applications in medicine and biotechnology. However, the mechanistic basis underlying the CRISPR-Cas9 function is unclear, preventing rational engineering of the system toward improved genome editing. We report on extensive molecular dynamics studies, including multi-microsecond and accelerated methods probing displacements over micro-to-milliseconds, characterizing the mechanism of conformational activation of Cas9, from its apo form up to the nucleic acid binding. These simulations disclosed a mechanism for RNA recruitment in which the domain relocations cause the formation of a positively charged cavity for nucleic acid binding. As well, we revealed the formation of a catalytically competent Cas9, which is prone for the catalysis of DNA. We showed how, upon DNA binding, the relocation of the catalytic HNH domain, assisted by conformational changes of the non-target DNA strand, activates Cas9 for catalysis. Finally, a mechanism of allosteric regulation, triggered by PAM binding and activating the catalytic domains for concerted catalysis has been discovered. Overall, our outcomes address the lack of mechanistic information on CRISPR-Cas9, improving our understanding of the activation process. This information is critical for structure-based design of the CRISPR-Cas9 system, which could impact the development of improved genome editing tools.