Journal
CELL
Volume 170, Issue 1, Pages 35-+Publisher
CELL PRESS
DOI: 10.1016/j.cell.2017.05.044
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Funding
- College of Natural Sciences Catalyst award
- CPRIT [R1214]
- Welch Foundation [F-1808]
- National Science Foundation [1453358]
- NIH [F32 AG053051, R01 GM120554]
- John Templeton Foundation [UTA16-000194]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1453358] Funding Source: National Science Foundation
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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 similar to 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.
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