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

Autor:	Jung C; Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA., Hawkins JA; Institute for Computational Engineering and Science, The University of Texas at Austin, Austin, TX 78712, USA., Jones SK Jr; Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA., Xiao Y; Department of Molecular Biology and Genetics, Cornell University, 253 Biotechnology Building, Ithaca, NY 14853, USA., Rybarski JR; Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA., Dillard KE; Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA., Hussmann J; Institute for Computational Engineering and Science, The University of Texas at Austin, Austin, TX 78712, USA., Saifuddin FA; Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA., Savran CA; School of Mechanical Engineering, Birck Nanotechnology Center, Purdue University, 1205 West State Street, West Lafayette, IN 47907, USA., Ellington AD; 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., Ke A; Department of Molecular Biology and Genetics, Cornell University, 253 Biotechnology Building, Ithaca, NY 14853, USA., Press WH; 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., Finkelstein IJ; 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.
Jazyk:	angličtina
Zdroj:	Cell [Cell] 2017 Jun 29; Vol. 170 (1), pp. 35-47.e13.
DOI:	10.1016/j.cell.2017.05.044
Abstrakt:	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 ∼10 7 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. (Copyright © 2017 Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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