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.
Original language | English |
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Pages (from-to) | 35-47.e13 |
Journal | Cell |
Volume | 170 |
Issue number | 1 |
DOIs | |
Publication status | Published - 2017 Jun 29 |
Externally published | Yes |
Bibliographical note
Publisher Copyright:© 2017 Elsevier Inc.
Keywords
- CRISPR
- Cas3
- Cascade
- biophysics
- fluorescence microscopy
- next generation sequencing
ASJC Scopus subject areas
- General Biochemistry,Genetics and Molecular Biology