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

作者：Jung Cheulhee; Hawkins John A; Jones Stephen K Jr; Xiao Yibei; Rybarski James R; Dillard Kaylee E; Hussmann Jeffrey; Saifuddin Fatema A; Savran Cagri A; Ellington Andrew D; Ke Ailong; Press William H; Finkelstein Ilya J

来源：Cell, 2017, 170(1): 35-+.

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 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.

• 出版日期2017-6-29