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

Publication date: 29 June 2017
Source:Cell, Volume 170, Issue 1
Author(s): 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
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

Graphical abstract

Teaser

Discarded next-gen sequencing chips provide a platform for analyzing protein-DNA interactions that reveals a novel proofreading mechanism used by the Cascade/Cas3 complex.


from # All Medicine by Alexandros G. Sfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2urHLHA
via IFTTT