Jung Cheulhee, Hawkins John A, Jones Stephen K, 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
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.
Institute for Computational Engineering and Science, The University of Texas at Austin, Austin, TX 78712, USA.
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 ∼10 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.
CRISPR-Cas核蛋白通过与crRNA碱基配对靶向外源DNA。然而，对于脱靶DNA序列上蛋白质结合和核酸酶激活的定量描述仍然难以捉摸。在此，我们描述了一种芯片杂交关联映射平台（CHAMP），该平台重新利用下一代测序芯片来同时测量蛋白质与约10个独特DNA序列之间的相互作用。使用CHAMP，我们首次全面研究了I-E型CRISPR-Cas（Cascade）复合物和Cas3核酸酶对DNA的识别。对突变靶序列和人类基因组DNA的分析表明，Cascade识别扩展的原间隔相邻基序（PAM）。Cascade以惊人的3个核苷酸周期性识别DNA。PAM和PAM近端核苷酸的身份通过释放Cse1亚基来控制Cas3的募集。这些发现被用于建立一个控制脱靶DNA结合的生物物理限制模型。CHAMP为高通量定量分析合成DNA和基因组DNA上的蛋白质-DNA相互作用提供了一个框架。回形针。
