The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation

Abstract
Data availability
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Abstract

CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional protein Cas4 has been implicated in selection and processing of prespacer substrates for Cas1-Cas2, although this mechanism remains unclear. We show that Cas4 interacts directly with Cas1-Cas2 forming a Cas4-Cas1-Cas2 complex that captures and processes prespacers prior to integration. Structural analysis of the Cas4-Cas1-Cas2 complex reveals two copies of Cas4 that closely interact with the two integrase active sites of Cas1, suggesting a mechanism for substrate handoff following processing. We also find that the Cas4-Cas1-Cas2 complex processes single-stranded DNA provided in cis or in trans with a double-stranded DNA duplex. Cas4 cleaves precisely upstream of PAM sequences, ensuring the acquisition of functional spacers. Our results explain how Cas4 cleavage coordinates with Cas1-Cas2 integration and defines the exact cleavage sites and specificity of Cas4.

Data availability

The negative-stain EM volumes for the Cas1-Cas2-target, asymmetrical Cas4-Cas1-Cas2-target, symmetrical Cas4-Cas1-Cas2-target, asymmetrical Cas4-Cas1-Cas2-prespacer and symmetrical Cas4-Cas1-Cas2-prespacer complexes have been deposited to EMDB under the accession numbers EMDB-20127, EMDB-20128, EMDB-20129, EMDB-20130 and EMDB-20131, respectively.

The following data sets were generated

(2019) Bacillus halodurans Cas1-Cas2-target
The Electron Microscopy Data Bank, EMDB-20127.

https://www.ebi.ac.uk/pdbe/emdb/
(2019) Bacillus halodurans Cas4-Cas1-Cas2-target asymmetrical
The Electron Microscopy Data Bank, EMDB-20128.

https://www.ebi.ac.uk/pdbe/emdb/
(2019) Bacillus halodurans Cas4-Cas1-Cas2-target symmetrical
The Electron Microscopy Data Bank, EMDB-20129.

https://www.ebi.ac.uk/pdbe/emdb/
(2019) Bacillus halodurans Cas4-Cas1-Cas2-prespacer asymmetrical
The Electron Microscopy Data Bank, EMDB-20130.

https://www.ebi.ac.uk/pdbe/emdb/
(2019) Bacillus halodurans Cas4-Cas1-Cas2-prespacer symmetrical
The Electron Microscopy Data Bank, EMDB-20131.

https://www.ebi.ac.uk/pdbe/emdb/

The following previously published data sets were used

(2014) Pyrobaculum calidifontis Cas4
Protein Data Bank, 4R5Q.

https://www.rcsb.org/structure/4R5Q
1. Kim TY
2. Shin M
3. Yen LHT
4. Kim JS
(2013) Crystal structure of Cas1 from Archaeoglobus fulgidus and its nucleolytic activity
Protein Data Bank, 4N06.

https://www.rcsb.org/structure/4N06
1. Ke A
2. Nam KH
(2012) Double-stranded Endonuclease Activity in B. halodurans Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas2 Protein
Protein Data Bank, 4ES3.

https://www.rcsb.org/structure/4ES3
(2014) Crystal structure of the Cas1-Cas2 complex from Escherichia coli
Protein Data Bank, 4P6I.

https://www.rcsb.org/structure/4P6I
(2015) Crystal structure the Escherichia coli Cas1-Cas2 complex bound to protospacer DNA
Protein Data Bank, 5DS4.

https://www.rcsb.org/structure/5DS4
1. Xiao Y
2. Ng S
3. Nam KH
4. Ke A
(2017) E. far Cas1-Cas2/prespacer binary complex
Protein Data Bank, 5XVN.

https://www.rcsb.org/structure/5XVN
1. Xiao Y
2. Ng S
3. Nam KH
4. Ke A
(2017) E. fae Cas1-Cas2/prespacer/target ternary complex revealing the fully integrated states
Protein Data Bank, 5XVP.

https://www.rcsb.org/structure/5XVP

Article and author information

Author details

Hayun Lee

Roy J Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, United States

Competing interests
The authors declare that no competing interests exist.
Yukti Dhingra

Roy J Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, United States

Competing interests
The authors declare that no competing interests exist.
Dipali G Sashital

Roy J Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, United States

For correspondence
sashital@iastate.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-7681-6987

Funding

National Institute of General Medical Sciences (GM115874)

Dipali G Sashital

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.