CRISPR Immunological Memory Requires a Host Factor for Specificity

James K Nuñez; Lawrence Bai; Lucas B Harrington; Tracey L Hinder; Jennifer A Doudna

doi:10.1016/j.molcel.2016.04.027

CRISPR Immunological Memory Requires a Host Factor for Specificity

Mol Cell. 2016 Jun 16;62(6):824-833. doi: 10.1016/j.molcel.2016.04.027. Epub 2016 May 19.

Authors

James K Nuñez¹, Lawrence Bai², Lucas B Harrington¹, Tracey L Hinder³, Jennifer A Doudna⁴

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
² Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
³ Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
⁴ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA 94720, USA; Center for RNA Systems Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Molecular Biophysics and Integrated Bioimaging, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address: doudna@berkeley.edu.

PMID: 27211867
DOI: 10.1016/j.molcel.2016.04.027

Abstract

Bacteria and archaea employ adaptive immunity against foreign genetic elements using CRISPR-Cas systems. To generate immunological memory, the Cas1-Cas2 protein complex captures 30-40 base pair segments of foreign DNA and catalyzes their integration into the host genome as unique spacer sequences. Although spacers are inserted strictly at the A-T-rich leader end of CRISPR loci in vivo, the molecular mechanism of leader-specific spacer integration remains poorly understood. Here we show that the E. coli integration host factor (IHF) protein is required for spacer acquisition in vivo and for integration into linear DNA in vitro. IHF binds to the leader sequence and induces a sharp DNA bend, allowing the Cas1-Cas2 integrase to catalyze the first integration reaction at the leader-repeat border. Together, these results reveal that Cas1-Cas2-mediated spacer integration requires IHF-induced target DNA bending and explain the elusive role of CRISPR leader sequences during spacer acquisition.

MeSH terms

Adaptive Immunity*
Binding Sites
CRISPR-Associated Proteins / genetics
CRISPR-Associated Proteins / immunology*
CRISPR-Associated Proteins / metabolism
CRISPR-Cas Systems / immunology*
Clustered Regularly Interspaced Short Palindromic Repeats / immunology*
DNA, Bacterial / chemistry
DNA, Bacterial / genetics
DNA, Bacterial / immunology*
DNA, Bacterial / metabolism
Endodeoxyribonucleases / genetics
Endodeoxyribonucleases / immunology*
Endodeoxyribonucleases / metabolism
Endonucleases / genetics
Endonucleases / immunology*
Endonucleases / metabolism
Escherichia coli / genetics
Escherichia coli / immunology*
Escherichia coli / metabolism
Escherichia coli Proteins / genetics
Escherichia coli Proteins / immunology*
Escherichia coli Proteins / metabolism
Immunologic Memory*
Integration Host Factors / genetics
Integration Host Factors / immunology*
Integration Host Factors / metabolism
Nucleic Acid Conformation
Protein Binding
Structure-Activity Relationship
Time Factors

Substances

CRISPR-Associated Proteins
DNA, Bacterial
Escherichia coli Proteins
Integration Host Factors
integration host factor, E coli
Cas2 protein, E coli
Endodeoxyribonucleases
Endonucleases
YgbT protein, E coli

Abstract

MeSH terms

Substances

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