Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays

Hayun Lee; Yi Zhou; David W Taylor; Dipali G Sashital

doi:10.1016/j.molcel.2018.03.003

Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays

Mol Cell. 2018 Apr 5;70(1):48-59.e5. doi: 10.1016/j.molcel.2018.03.003. Epub 2018 Mar 27.

Authors

Hayun Lee¹, Yi Zhou², David W Taylor³, Dipali G Sashital⁴

Affiliations

¹ Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA.
² Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
³ Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, USA.
⁴ Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA, USA. Electronic address: sashital@iastate.edu.

Abstract

CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specific endonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration.

Keywords: CRISPR-Cas; Cas1-Cas2; Cas4; DNA cleavage; DNA integration; negative stain EM.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

CRISPR-Associated Protein 9 / genetics*
CRISPR-Associated Protein 9 / immunology
CRISPR-Associated Protein 9 / isolation & purification
CRISPR-Associated Protein 9 / metabolism
CRISPR-Associated Proteins / genetics*
CRISPR-Associated Proteins / immunology
CRISPR-Associated Proteins / metabolism
CRISPR-Cas Systems*
Clustered Regularly Interspaced Short Palindromic Repeats*
DNA, Bacterial / genetics*
DNA, Bacterial / immunology
DNA, Bacterial / metabolism
Endodeoxyribonucleases / genetics
Endodeoxyribonucleases / metabolism
Escherichia coli / enzymology
Escherichia coli / genetics*
Escherichia coli Proteins / genetics
Escherichia coli Proteins / metabolism
Gene Editing / methods*
Models, Molecular
Multienzyme Complexes
Nucleic Acid Conformation
Protein Conformation
Protein Subunits
Substrate Specificity

Substances

CRISPR-Associated Proteins
DNA, Bacterial
Escherichia coli Proteins
Multienzyme Complexes
Protein Subunits
CRISPR-Associated Protein 9
Endodeoxyribonucleases
YgbT protein, E coli

Grants and funding

R01 GM115874/GM/NIGMS NIH HHS/United States