核酸等温扩增技术在临床和现场快速检测领域的应用前景广泛。然而，目前的核酸等温扩增技术仍存在如不完全等温、引物设计繁琐和拓展性较差等缺陷，新技术的开发因此具有重要意义。申请人近期构建的CRISDA技术是一种针对双链核酸的CRISPR等温扩增技术。其在灵敏度、特异性、简便性和普适性方面具有独特优势。同时，该技术的性能仍有进一步提升的空间。因此，本项目拟对该CRISPR等温扩增体系的机理和优化展开深入研究，进一步提升其扩增效率和特异性。具体研究内容包括：一，通过SpyCas9蛋白HNH结构域的工程化改造，降低其空间位阻对扩增反应的影响，从而优化扩增反应起始效率；二，通过发掘新型热稳定Cas9，构建高温扩增反应体系，并结合HNH结构域改造，实现扩增反应效率和特异性的进一步提升。本项目将有助于推动CRISPR技术在基因检测领域的发展，并为完善Cas9工程化改造策略和拓展CRISPR工具箱奠定基础。

Nucleic acids isothermal amplification techniques have exhibited great potentials in field testing and clinic applications. However, due to limitations such as the requirements of heat denaturation, complicated primer design, and poor versatility, the development of novel isothermal DNA amplification/detection method is highly desirable. CRISPR/Cas9-triggered nicking endonuclease-mediated strand displacement amplification method (abbreviated as CRISDA), developed by the applicant recently, is an ultra-sensitive and true isothermal approach for the amplification and detection of double-stranded DNA. Although CRISDA technique possesses distinctive advantages in sensitivity, specificity, simplicity and versatility, further improvements are feasible. Thus, in this project, we aim to systematically investigate underlying mechanisms of this CRISPR-based isothermal amplification method, explore potential optimization strategies, and eventually enhance its performances. Research will be focused on: 1. optimizing the HNH domain in SpyCas9 to reduce corresponding steric hindrance, and improve the amplification efficiency; and 2. exploring novel thermal stable Cas9 proteins to establish high-temperature CRISPR-based isothermal amplification reactions for further improved efficiency and specificity. This research may not only promote the development of CRISPR-based diagnosis approaches, but also provide fundamental insights for engineering Cas9 proteins and expanding the CRISPR toolbox.