发展基于人工核酸酶的条件性基因突变技术研究在体细胞迁移

Cell migration is an essential feature of metazoan development. Studies from in vitro cultured cell lines provide a conceptual framework in understanding cell migration. However, in vitro system cannot fully mimic complicated extracellular environment in vivo. It is thus important to study the dynamic behavior of cell migration in living animals. Many key regulators of cell migration are involved in various cellular developmental processes and their mutations often cause embryonic lethality, precluding the analysis of their contribution in cell migration during the later stage of embryonic development or postembryonic stage. The development of an efficient and convenient conditional knockout technique is highly demanded. Our recent work reported a novel strategy of using engineered nuclease, TALEN, to achieve conditional targeted genomic mutations in the model organism C. elegans. Our preliminary work has developed a novel conditional knockout technique based on another engineered CRISPR-Cas9, which is easier to manipulate than TALEN. We have also obtained higher mutation frequency of multiple genes using CRISPR-Cas9. This proposal will combine somatic CRISPR-Cas9 and TALEN techniques to study C. elegans Q neuroblast migration. We aim to understand the potential roles of embryonic lethal genes in in cell migration, and we will further apply our multiple-dimensional fluorescent live imaging technology to address the underlying cellular mechanism of these molecules in cell migration in live C. elegans. We expect that our study can provide new insights into the development and therapy of cell migration related diseases.

细胞迁移是多细胞生物体的重要特征。来自体外培养细胞的研究为理解细胞迁移提供基本理论框架，但体外系统难以模拟复杂的胞外环境，在活体动物中开展细胞迁移动态的研究是该领域的重要方向。调控细胞迁移的许多关键因子参与多种生物学过程，其突变导致胚胎致死，发展高效便捷的条件性基因敲除技术成为研究在体细胞迁移的迫切需要。申请人已发表在模式动物线虫中建立了基于人工核酸内切酶TALEN的条件性基因突变方法。以此为基础，本课题的前期工作发展了基于人工核酸内切酶CRISPR-Cas9的条件性基因突变技术，在多个基因上获得了比TALEN技术更高的条件性突变效率。本课题拟选择线虫Q神经前体细胞的迁移为对象，结合体细胞CRISPR-Cas9和TALEN条件性基因突变技术，研究调控胚胎发育的关键因子在细胞迁移的可能作用，利用多维荧光活体显微成像技术揭示其调控细胞迁移的机制，为理解细胞迁移相关疾病的发生和治疗提供新思路。

正向和反向遗传学手段在秀丽隐杆线虫中已经得到大规模地运用，然而，在秀丽隐杆线虫中高效制备条件性突变体的手段仍亟待发展。本课题通过控制人工核酸内切酶CRISPR-Cas9在体细胞中的时空表达实现基因的条件性突变，我们发现体细胞CRISPR-Cas9系统在多个基因上获得了比体细胞TALEN 技术更高的条件性突变效率，而且体细胞CRISPR-Cas9系统可以同时突变多个基因，让我们能够快速便捷地制备条件性双突体或者三突体。我们利用体细胞CRISPR-Cas9系统制备了一系列胚胎发育必需的细胞骨架基因在线虫Q 神经前体细胞的条件性突变品系，研究胚胎关键基因调控胚胎后发育过程中的神经前体细胞迁移的分子细胞学机制。本课题发现1）与人类神经功能紊乱相关的Coronin基因在线虫Q神经前体细胞迁移过程中调控微丝骨架结构和细胞形态；2）在Q神经前体细胞迁移过程中，跨膜蛋白MIG-13/Lrp12分别与ABL-1和SEM-5/Grb2直接结合，从而把信号传递到微丝成核促进因子WAVE和WASP上，最终激活Arp2/3复合物。WAVE的缺失会破化迁移Q细胞的微丝结构并减少Q细胞的迁移，WASP的突变不会破坏Q神经前体细胞迁移，但可以增强WAVE缺失导致的迁移缺陷。因此MIG-13-ABL-1-WAVE是为Q细胞迁移提供动力的主要通路，MIG-13-SEM-5-WASP通路可以部分补偿WAVE通路的缺失，两条信号通路协同作用调控Q神经前体细胞的迁移。本课题研究成果揭示了细胞迁移的新调控机制，为理解人类细胞迁移相关疾病发生机制提供新思路。