莱茵衣藻的高通量基因组编辑技术研究

Eukaryotic microalgae have significant economic value. Genetic reconstruction of microalgal genomes is helpful to obtain high yield and quality of algal strains for industrial production. But there are many limitations for the existing methods of genetic manipulation of microalgae. Therefore, it is urgent to establish an efficient means of genetic engineering for microalgae. Chlamydomonas reinhardtii known as “green yeast” is widely used as a class of economic microalgae and is also an important model for research in cell life. In this study, expression vectors of Cas9 protein and sgRNA will be constructed based on genetic transformation system of C. reinhardtii for the first time. An efficiently targeted genome editing technique of C. reinhardtii will be developed to achieve knockout, knockin and mutations of the targeted gene. High-throughput editing on the whole genome will be performed through the design and synthesis of targeted sequence libraries for C. reinhardtii genome and construction of targeted vector libraries. Targeted mutant library of C. reinhardtii will then be obtained. Cells with a specific phenotype will be selected by flow cytometry from the mutant library. High-throughput sequencing method of sgRNA within the cell population will be established to identify genes closely related to the phenotype. In summary, a high-throughput genome editing technique of C. reinhardtii is expected to be developed for the first time in this project and will promote the study of genetic and metabolic regulatory mechanisms of microalgae. It will also accelerate the pace of genetic engineering of microalgae and have important theoretical implications and technology value for the development of microalgae industry.

真核微藻具有重要的经济价值，对微藻基因组进行遗传改造有助于获得优质高产的工业化藻种，但现有的微藻遗传操作方法十分局限，亟须建立一种高效的基因工程手段。莱茵衣藻被称为绿色酵母，是一类应用广泛的经济微藻，也是研究细胞生命活动的重要模式生物。本研究将基于莱茵衣藻的遗传转化体系构建Cas9蛋白和sgRNA的表达载体，建立高效的莱茵衣藻基因组定向编辑技术，实现对目标基因的定向敲除、敲入和突变。通过设计和合成针对莱茵衣藻基因组的靶向序列库，构建靶向载体库，在全基因组水平上进行高通量编辑，获得莱茵衣藻定向突变体库。采用流式细胞仪从突变体库中分选具有特定表型的单细胞群，建立细胞群体内sgRNA的高通量测序方法，用以鉴定与该表型密切相关的基因。本项目首次建立的莱茵衣藻高通量基因组编辑技术，将会促进微藻遗传和代谢调控机制的研究，加速微藻基因工程改造的步伐，对微藻产业的发展具有重要的理论指导意义和技术应用价值。

莱茵衣藻是研究细胞生命活动的重要模式生物，本研究基于莱茵衣藻的遗传转化体系，开展了基因编辑载体的构建和衣藻转化实验，构建了Cas9基因、Cpf1基因与gRNA的复合表达载体，采用多种方法进行衣藻细胞转化，未检测到靶向载体完全整合的转化子，针对4个衣藻基因的靶向敲出也未能得到高效率的基因编辑突变体。同时进行了Cas9蛋白与gRNA复合体的转化实验，得到的衣藻转化子中未检测到突变体，这一结果与文献报道的极低的基因编辑效率一致。对非同源末端修复的关键基因Ku70和Ku80进行了表达抑制，以期提高同源重组效率，用构建的maa7基因重组片段转化末端修复抑制的衣藻细胞，但未能得到重组突变体。总之，本项目研究过程中发现衣藻细胞与现有的基因编辑蛋白存在一定的排斥性，导致基因编辑效率难以提高。目前的工作正在进行Cpf1蛋白与gRNA复合体的衣藻转化实验，后续将尝试更多不同来源的基因编辑蛋白。