Structural and Dynamics Studies of the Spcas9 Variant Provide Insights into the Regulatory Role of the REC1 Domain

Huayi Liu, Yi Zhou, Yingjie Song, Qianqian Zhang, Yeyi Kan, Xinyue Tang, Qingjie Xiao, Qianyin Xiang, Huanxiang Liu, Yunzi Luo, Rui Bao

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

CRISPR-Cas9 from Streptococcus pyogenes has been harnessed as a powerful biotechnological tool for DNA sequence modification. The gRNA binding the recognition lobe (REC lobe) of Cas9 is the initial step of Cas9-mediated DNA cleavage and is a prerequisite to trigger the subsequent conformational transition of Cas9. The functions of REC1-3 domains have been summarized as "sense" nucleic acids, "regulate" the HNH conformational transition, and finally "lock" the HNH domain at the cleavage site. Therefore, understanding the dynamic mechanism of the REC lobe is important to design and engineer better Cas9 enzymes. Here, we solved the apo form structure of a SpCas9 variant (xCas9 P411T) that harbors seven mutations in the SpCas9 REC region and one mutation in the C-terminal domain. This structure reveals that the mutations in the REC lobe resulted in a distinct conformation of the REC1 domain. Together with the biochemical results, we hypothesized that the dynamic properties of the REC1 domain are associated with Cas9 PAM specificity. Furthermore, using molecular dynamics simulations to model the conformational activation process of xCas9 P411T, we found that REC1 acts as a hub that modulates multiple domain motion during Cas9 activation and target site binding. These results lay the foundation for engineering an improved CRISPR-Cas9 system.

Original languageEnglish
Pages (from-to)8687-8697
Number of pages11
JournalACS Catalysis
Volume12
Issue number14
DOIs
Publication statusPublished - 15 Jul 2022

Keywords

  • SpCas9
  • conformational activation
  • crystal structure
  • molecular dynamics simulation
  • recognition domain 1 (REC1)

Fingerprint

Dive into the research topics of 'Structural and Dynamics Studies of the Spcas9 Variant Provide Insights into the Regulatory Role of the REC1 Domain'. Together they form a unique fingerprint.

Cite this