斑马鱼中原始生殖细胞靶向的高效基因敲除和敲入技术建立

As a new genome editing system, CRISPR/Cas9 could cut specific DNA sequences, and then mediate subsequent knock-out or knock-in. However, the cutting efficiency of this system varies at different genomic loci, and it will expend a lot of energy on screening of knock-out or knock-in with low efficiency. Besides, when targeting the essential genes of early development, embryo survival rate would conflict with germ cell mutation rate. We would establish highly efficient knock-out and knock-in in zebrafish on primordial germ cells (PGCs) manipulation. Firstly, we would construct PGC-targeted Cas9 expression transgenic lines. In comparison with the common knock-out on whole embryo, it would be more effective to establish the mutants targeting the genomic locus with low binding efficiency and the essential genes of early development by the transgenic lines. Then, using the embryo of PGC-targeted Cas9 expression transgenic lines, and the donor vector based on microhomology-mediated end joining (MMEJ) repair system for screening embryos carrying knock-in PGCs, we would generate knock-in line targeted endogenous gene. Our strategy would substantially improve the knock-out and knock-in efficiency in germ cell, providing more powerful tools for generating zebrafish new model.

作为一种新型的基因组编辑工具，CRISPR/Cas9能够在基因组特定位点产生DNA双链断裂，从而介导后续的基因敲除或敲入。然而，该系统对基因组不同座位的剪切效率存在巨大差异，对一些低效靶位进行敲除或敲入时面临巨大的筛选工作量；此外，在对早期发育必需基因进行敲除时，胚胎存活率和生殖传递率往往成为不可调和的一对矛盾。本项目拟在斑马鱼中建立原始生殖细胞（PGC）靶向的高效基因敲除和敲入技术。首先，构建PGC高效、特异表达Cas9的转基因系。相比于传统的全胚水平的敲除技术，利用该品系将极大有助于低效靶位和早期发育必需基因的突变体获得。随后，将转基因品系作为受体鱼，利用基于微同源末端连接（MMEJ）介导的DNA双链断裂修复敲入载体对PGC发生插入事件的胚胎进行早期筛选，构建出靶向内源基因的敲入品系。本项目研究将大幅提高生殖细胞中的基因敲除和敲入效率，为斑马鱼工具品系的研制提供更加高效的技术手段。

新一代人工核酸内切酶技术——CRISPR/Cas9基因编辑技术的出现彻底改变了许多实验动物中的反向遗传方法。虽然目前利用CRISPR/Cas9基因编辑技术可以在斑马鱼中高效制备简单的插入或缺失突变，但是斑马鱼中实现精确的基因敲入或者序列编辑，对于大多数位点仍然较难实现。本研究首先筛选出在斑马鱼胚胎中产生特异高效的核酸内切酶，并以该核酸内切酶为工具，构建了斑马鱼胚胎泛表达Cas9的斑马鱼转基因品系，以及在PGC特异、高效、持续表达Cas9的转基因品系。同时，建立了PGC靶向的早期筛选系统，利用该系统能够在胚胎早期鉴别生殖细胞被有效操作的胚胎。进一步， 本研究利用CRISPR/Cas9系统和和细胞修复机制将外源基因整合到靶基因座中，建立了通过人为设计敲入载体上的gRNA靶点与基因组靶点微同源，在内源基因中能产生高效整合的基因敲入技术。