对基因功能全面清晰的阐释是后基因组时代的重要研究内容，它很大程度依赖于DNA高效编辑技术和工具的发明和运用。CRISPR/Cas系统作为近年最新发展起来的高效便捷DNA编辑技术，已经在许多生物中得到了成功的验证，并展现出了巨大的应用潜力。然而，其脱靶问题却成为制约其深入应用的重要瓶颈。尽管人们正试图通过各种方法来克服其脱靶效应，但至今如何降低脱靶率、提高其靶向特异性仍是这一领域尚未完全解决的一个关键科学问题。因此，本申请项目拟以CRISPR/Cas系统中目前应用最为广泛、来自Streptococcus pyogenes的CRISPR/Cas9为研究对象，综合运用体外和体内实验体系，并结合已知的Cas9晶体结构信息，系统考察PAM和sgRNA的相对空间位置等因素对Cas9脱靶产生的影响，并试图阐明其靶向特异性的分子机制，为人们更透彻地理解其作用机理，并为最终解决脱靶问题提供新的思路和策略。

A comprehensive and clear elucidation of gene function is one of most important tasks in the post-genome era. It relies heavily on the invention and use of highly efficient DNA editing tools. CRISPR/Cas as a rapidly developing technology for DNA editing has been successfully validated in different kinds of organisms and has shown its great potential for various applications. However, the off-target could be a bottleneck of its further utilization. Currently, how to reduce the off-target and increase targeting specificity of CRISPR/Cas is still a key issue which has not been fully solved although researchers are trying to overcome this problem through various methods. Thus, we propose here to undertake work on comprehensive investigation of impacts of relative position of PAM and sgRNA on off-target by means of in vitro and in vivo, combined with known crystal information, using the most widely applied Cas9 from Streptococcus pyogenes as subject. As a result, we attempt to clarify the molecular mechanism of its targeting specificity to provide an alternative strategy for better understanding of off-target and finally solving this problem.