CRISPR-Cas13a系统天然抑制蛋白AcrVIA1的鉴定与分子机制研究

CRISPR-Cas系统构成了原核生物适应性免疫防御系统，已发展成为强有力的基因编辑工具。为了克服CRISPR免疫，噬菌体编码anti-CRISPR（Acr）蛋白来抑制CRISPR系统，从而为基于CRISPR的应用提供了有效的“开关”工具。目前，包括Cas9、Cpf1在内的多个CRISPR系统的Acr蛋白相继被发现，而Cas13a（C2c2）系统的Acr蛋白至今仍未被报道。在本研究的前期工作中，我们通过生物信息学方法，初步确立了Cas13a系统的候选Acr蛋白。并利用大肠杆菌筛选体系成功发现一类可抑制Leptotrichia wadei菌种Cas13a的Acr蛋白，已命名为AcrVIA1。在此基础上，我们将进一步探究其在体外以及人类细胞内对Cas13a的抑制活性。另外，我们将筛选CRISPR-Cas13a系统其他AcrVIA蛋白，并最终阐明AcrVIA蛋白抑制Cas13a系统的分子机制。

CRISPR-Cas systems serve as the adaptive immune system for prokaryotes and have been used as a powerful tool for genome editing. To overcome CRISPR immunity, phages encode anti-CRISPR proteins (Acrs) to inhibit CRISPR systems, providing an efficient "off-switch" tool for CRISPR-based applications. So far, Acr proteins of multiple CRISPR-Cas systems, including Cas9 and Cpf1, have been discovered. However, Acr proteins of Cas13a (C2c2) system have not been reported. In the preliminary work of this study, we have initially established candidate Acr proteins of the Cas13a system through bioinformatics methods. Using the E. coli screening system, a family of Acr proteins that can inhibit Cas13a derived from Leptotrichia wadei strain (LwaCas13a) was successfully identified and then named AcrVIA1. On this basis, we will further investigate its inhibitory activities on Cas13a in vitro and in human cells. In addition, we will screen other AcrVIA proteins of the CRISPR-Cas13a system, and finally clarify the molecular mechanism of AcrVIA protein in Cas13a inhibition.