Type IV-A CRISPR-Csf complex: Assembly, dsDNA targeting, and CasDinG recruitment

Ning Cui; Jun-Tao Zhang; Yongrui Liu; Yanhong Liu; Xiao-Yu Liu; Chongyuan Wang; Hongda Huang; Ning Jia

doi:10.1016/j.molcel.2023.05.036

Type IV-A CRISPR-Csf complex: Assembly, dsDNA targeting, and CasDinG recruitment

Mol Cell. 2023 Jul 20;83(14):2493-2508.e5. doi: 10.1016/j.molcel.2023.05.036. Epub 2023 Jun 20.

Authors

Ning Cui¹, Jun-Tao Zhang¹, Yongrui Liu², Yanhong Liu², Xiao-Yu Liu¹, Chongyuan Wang³, Hongda Huang⁴, Ning Jia⁵

Affiliations

¹ Department of Biochemistry, School of Medicine, Southern University of Science and Technology, Shenzhen 518055, China.
² Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes, Department of Chemical Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, China.
³ Faculty of Pharmaceutical Sciences, Shenzhen Institutes of Advanced Technology, Chinese Academy of Science, Shenzhen 518055, China; Center for Human Tissues and Organs Degeneration, Shenzhen Institutes of Advanced Technology, Chinese Academy of Science, Shenzhen 518055, China.
⁴ Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes, Department of Chemical Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, China. Electronic address: huanghd@sustech.edu.cn.
⁵ Department of Biochemistry, School of Medicine, Southern University of Science and Technology, Shenzhen 518055, China; Shenzhen Key Laboratory of Cell Microenvironment, Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Southern University of Science and Technology, Shenzhen 518055, China; Key University Laboratory of Metabolism and Health of Guangdong, Southern University of Science and Technology, Shenzhen 518055, China. Electronic address: jian@sustech.edu.cn.

PMID: 37343553
DOI: 10.1016/j.molcel.2023.05.036

Abstract

Type IV CRISPR-Cas systems, which are primarily found on plasmids and exhibit a strong plasmid-targeting preference, are the only one of the six known CRISPR-Cas types for which the mechanistic details of their function remain unknown. Here, we provide high-resolution functional snapshots of type IV-A Csf complexes before and after target dsDNA binding, either in the absence or presence of CasDinG, revealing the mechanisms underlying Csf^crRNA complex assembly, "DWN" PAM-dependent dsDNA targeting, R-loop formation, and CasDinG recruitment. Furthermore, we establish that CasDinG, a signature DinG family helicase, harbors ssDNA-stimulated ATPase activity and ATP-dependent 5'-3' DNA helicase activity. In addition, we show that CasDinG unwinds the non-target strand (NTS) and target strand (TS) of target dsDNA from the Csf^crRNA complex. These molecular details advance our mechanistic understanding of type IV-A CRISPR-Csf function and should enable Csf complexes to be harnessed as genome-engineering tools for biotechnological applications.

Keywords: CRISPR; CasDinG recruitment; PAM recognition; R-loop formation; bacterial defense systems; crRNA maturation; dsDNA targeting; genome engineering; helicase activity; type IV-A CRISPR-Csf system.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Associated Proteins* / metabolism
CRISPR-Cas Systems
DNA* / genetics
DNA, Single-Stranded / genetics

Substances

DNA
DNA, Single-Stranded
CRISPR-Associated Proteins