DNA Targeting by a Minimal CRISPR RNA-Guided Cascade

Megan L Hochstrasser; David W Taylor; Jack E Kornfeld; Eva Nogales; Jennifer A Doudna

doi:10.1016/j.molcel.2016.07.027

DNA Targeting by a Minimal CRISPR RNA-Guided Cascade

Mol Cell. 2016 Sep 1;63(5):840-51. doi: 10.1016/j.molcel.2016.07.027.

Authors

Megan L Hochstrasser¹, David W Taylor², Jack E Kornfeld³, Eva Nogales⁴, Jennifer A Doudna⁵

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
² Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address: dtaylor@utexas.edu.
³ Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
⁴ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Molecular Biophysics and Integrative Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
⁵ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA; Molecular Biophysics and Integrative Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address: doudna@berkeley.edu.

Abstract

Bacteria employ surveillance complexes guided by CRISPR (clustered, regularly interspaced, short palindromic repeats) RNAs (crRNAs) to target foreign nucleic acids for destruction. Although most type I and type III CRISPR systems require four or more distinct proteins to form multi-subunit surveillance complexes, the type I-C systems use just three proteins to achieve crRNA maturation and double-stranded DNA target recognition. We show that each protein plays multiple functional and structural roles: Cas5c cleaves pre-crRNAs and recruits Cas7 to position the RNA guide for DNA binding and unwinding by Cas8c. Cryoelectron microscopy reconstructions of free and DNA-bound forms of the Cascade/I-C surveillance complex reveal conformational changes that enable R-loop formation with distinct positioning of each DNA strand. This streamlined type I-C system explains how CRISPR pathways can evolve compact structures that retain full functionality as RNA-guided DNA capture platforms.

MeSH terms

Amino Acid Motifs
Bacterial Proteins / chemistry
Bacterial Proteins / genetics*
Bacterial Proteins / metabolism
Binding Sites
CRISPR-Cas Systems*
Cloning, Molecular
Cryoelectron Microscopy
DNA / chemistry
DNA / genetics*
DNA / metabolism
Desulfovibrio vulgaris / genetics*
Desulfovibrio vulgaris / metabolism
Endonucleases / chemistry
Endonucleases / genetics*
Endonucleases / metabolism
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Editing
Gene Expression
Kinetics
Models, Molecular
Nucleic Acid Conformation
Operon
Protein Binding
Protein Conformation, alpha-Helical
Protein Interaction Domains and Motifs
RNA, Bacterial / chemistry
RNA, Bacterial / genetics*
RNA, Bacterial / metabolism
RNA, Guide, CRISPR-Cas Systems / chemistry
RNA, Guide, CRISPR-Cas Systems / genetics*
RNA, Guide, CRISPR-Cas Systems / metabolism
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Substrate Specificity

Substances

Bacterial Proteins
RNA, Bacterial
RNA, Guide, CRISPR-Cas Systems
Recombinant Proteins
DNA
Endonucleases

Abstract

MeSH terms

Substances

Grants and funding