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

Cheulhee Jung, John A. Hawkins, Stephen K. Jones, 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

Research output: Contribution to journalArticle

36 Citations (Scopus)

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 [Figure presented]

Original languageEnglish
Pages (from-to)35-47.e13
JournalCell
Volume170
Issue number1
DOIs
Publication statusPublished - 2017 Jun 29
Externally publishedYes

Keywords

  • biophysics
  • Cas3
  • Cascade
  • CRISPR
  • fluorescence microscopy
  • next generation sequencing

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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  • Cite this

    Jung, C., Hawkins, J. A., Jones, S. K., Xiao, Y., Rybarski, J. R., Dillard, K. E., Hussmann, J., Saifuddin, F. A., Savran, C. A., Ellington, A. D., Ke, A., Press, W. H., & Finkelstein, I. J. (2017). Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on Next Generation Sequencing Chips. Cell, 170(1), 35-47.e13. https://doi.org/10.1016/j.cell.2017.05.044