Crispr/Cas3解旋和降解DNA分子机制研究

CRISPR is prokaryotic adaptive immune system, using RNA interference to degrade invasive pathogenic nucleic acid, also widely used as an important genome editing tool. CRISPR application requires efficient and stable Cas3, the structural biological research on it will help to elucidate its mechanism and guide the molecular modification of native Cas3, having great value in theory and application. Recently, we determined the complex structure of Cas3 bound to DNA substrate (NSMB, 2014), reporting the structural basis of Cas3 binding DNA substrate, and revealing the mechanism of Cas3 unwinding and degrading foreign DNA. Although this scientific breakthrough preliminarily clarified its mechanism, the limitation of DNA substrate and lack of homologous structure prevent our understanding of its structural details. The further research in this project aims to determine the crystal structure of Cas3 bound to DNA substrate with specific structure and structures of homologous Cas3. These structures will reveal the key amino acid that unwinding DNA and detailed Cas3-DNA interaction; meanwhile, the solution and comparison of homologous structures will show Cas3 core structure elements. This research will provide the structural biology basis for CRISPR mechanism and application.

CRISPR是原核生物利用RNA干扰降解核酸、抵御病原入侵的获得性免疫系统，也作为重要的基因组编辑工具得到广泛应用。CRISPR的应用需要高效、稳定的Cas3，对其结构生物学的研究有助于阐明其作用机制、指导天然Cas3的分子改造，具有重要的理论和应用价值。申请人最近解析了Cas3-DNA的复合物结构(NSMB, 2014)，报道了Cas3结合DNA底物的结构基础，并揭示了Cas3解旋和降解入侵DNA的机制。这一学术突破虽然初步阐明了其作用机制，但是DNA底物的局限性和同源结构的匮乏阻碍了对其结构细节的理解。在此基础申请人提出本项申请，拟通过晶体学的方法来解析Cas3结合特殊构造DNA底物的结构和Cas3的同源结构，揭示解旋DNA的关键氨基酸以及精确的蛋白-DNA相互作用，同时通过同源结构的解析和比对得到Cas3的核心构成元件，为CRISPR系统的机制研究和应用提供结构生物学基础。