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.


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*


  • DNA-Binding Proteins