Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips

Cheulhee Jung; John A Hawkins; Stephen K Jones Jr; Yibei Xiao; James R Rybarski; Kaylee E Dillard; Jeffrey Hussmann; Fatema A Saifuddin; Cagri A Savran; Andrew D Ellington; Ailong Ke; William H Press; Ilya J Finkelstein

doi:10.1016/j.cell.2017.05.044

Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips

Cell. 2017 Jun 29;170(1):35-47.e13. doi: 10.1016/j.cell.2017.05.044.

Affiliations

¹ Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.
² Institute for Computational Engineering and Science, The University of Texas at Austin, Austin, TX 78712, USA.
³ Department of Molecular Biology and Genetics, Cornell University, 253 Biotechnology Building, Ithaca, NY 14853, USA.
⁴ School of Mechanical Engineering, Birck Nanotechnology Center, Purdue University, 1205 West State Street, West Lafayette, IN 47907, USA.
⁵ Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX 78712, USA.
⁶ Institute for Computational Engineering and Science, The University of Texas at Austin, Austin, TX 78712, USA; Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA; Department of Integrative Biology, The University of Texas at Austin, Austin, TX 78712, USA.
⁷ Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA; Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX 78712, USA. Electronic address: ifinkelstein@cm.utexas.edu.

Abstract

CRISPR-Cas nucleoproteins target foreign DNA via base pairing with a crRNA. However, a quantitative description of protein binding and nuclease activation at off-target DNA sequences remains elusive. Here, we describe a chip-hybridized association-mapping platform (CHAMP) that repurposes next-generation sequencing chips to simultaneously measure the interactions between proteins and ∼10⁷ unique DNA sequences. Using CHAMP, we provide the first comprehensive survey of DNA recognition by a type I-E CRISPR-Cas (Cascade) complex and Cas3 nuclease. Analysis of mutated target sequences and human genomic DNA reveal that Cascade recognizes an extended protospacer adjacent motif (PAM). Cascade recognizes DNA with a surprising 3-nt periodicity. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment by releasing the Cse1 subunit. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding. CHAMP provides a framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA. PAPERCLIP.

Keywords: CRISPR; Cas3; Cascade; biophysics; fluorescence microscopy; next generation sequencing.

MeSH terms

CRISPR-Cas Systems
DNA-Binding Proteins / analysis*
Electrophoretic Mobility Shift Assay
High-Throughput Nucleotide Sequencing / methods*
Microscopy, Fluorescence
Nucleotide Motifs
Protein Binding*
Sequence Analysis, DNA / methods*

Substances

DNA-Binding Proteins

Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips

Authors

Affiliations

Abstract

MeSH terms

Substances

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