Format

Send to

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

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

Author information

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

PMID:
28666121
PMCID:
PMC5552236
DOI:
10.1016/j.cell.2017.05.044
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center