@article{d33b041a6e544878bbfe72b2cf1c5245,
title = "Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips",
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.",
keywords = "CRISPR, Cas3, Cascade, biophysics, fluorescence microscopy, next generation sequencing",
author = "Cheulhee Jung and Hawkins, {John A.} and Jones, {Stephen K.} and Yibei Xiao and Rybarski, {James R.} and Dillard, {Kaylee E.} and Jeffrey Hussmann and Saifuddin, {Fatema A.} and Savran, {Cagri A.} and Ellington, {Andrew D.} and Ailong Ke and Press, {William H.} and Finkelstein, {Ilya J.}",
note = "Funding Information: We are indebted to our colleague Scott Hunicke-Smith and members of the Genomic Sequencing and Analysis Facility for valuable insights. We are grateful to members of the Finkelstein laboratory for carefully reading the manuscript. This work was supported by a College of Natural Sciences Catalyst award, CPRIT (R1214 to I.J.F.), the Welch Foundation (F-1808 to I.J.F.), the National Science Foundation (1453358), and the NIH (F32 AG053051 to S.K.J. and R01 GM120554 to I.J.F.). This publication was also made possible through the support of a grant from the John Templeton Foundation (UTA16-000194 to A.D.E). The opinions expressed in this publication are those of the authors and do not necessarily reflect the views of the John Templeton Foundation. I.J.F. is a CPRIT Scholar in Cancer Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Science Foundation. Publisher Copyright: {\textcopyright} 2017 Elsevier Inc.",
year = "2017",
month = jun,
day = "29",
doi = "10.1016/j.cell.2017.05.044",
language = "English",
volume = "170",
pages = "35--47.e13",
journal = "Cell",
issn = "0092-8674",
publisher = "Cell Press",
number = "1",
}