脊尾白虾胞苷和腺苷脱氨酶作为单碱基基因编辑工具酶的可行性研究

Crustaceans are important species of marine aquaculture and play an important role in marine fisheries. The study of gene function is helpful to explain the law of life at the molecular level. Researchers have found a large number of SNP sites in the genome of crustacean animals. It is difficult to study the function of genes through single-nucleotide mutagenesis in higher species. The single base editing has been used to study gene function in some species. However, there is no report in crustaceans at present. In our previous research, we firstly reported site-specific genome editing in Exopalaemon carinicauda by CRISPR/Cas9 technology. In this project, we will firstly construct a single base editing system in E. carinicauda based on the endogenous cytidine deaminase (CDA) and adenosine deaminase (ADA) (named EcCDA and EcADA) and nCas9(D10A) to carry out the conversions of different bases (C→T, G→A, A→G, T→C) on genomic DNA. Then, the feasibility of constructed single base editing system will be tested in vitro and in vivo. Next, the chitin synthesis gene from E. carinicauda (EcCHS) will be used as the target gene to furtherly assess the feasibility of EcCDA and EcADA in single base editing of E. carinicauda. By this system, the bases encoding the important amino acids in the typical Chitin_synth_2 domains of EcCHS are substituted to produce mutant prawns. The mutant and wild-type prawns are used to clarify the functions of EcCHS in the physiological and biochemical processes including the reproductive behavior, growth and molting, etc. The results of this project will not only be helpful to promote the study of functional genome of E. carinicauda, but also accelerate broad use of the single base editing in other crustaceans.

基于胞苷脱氨酶（CDA）和腺苷脱氨酶（ADA）的单碱基编辑技术在甲壳动物中未见报道。本项目拟以脊尾白虾为研究对象，在项目组实现了CRISPR/Cas9基因编辑基础上，利用内源CDA和ADA（EcCDA和EcADA）与Cas9突变体nCas9(D10A)，构建基于EcCDA和EcADA的单碱基编辑系统pEcCBE和pEcABE，实现对其基因组DNA不同碱基的替换（C→T，G→A，A→G，T→C），并通过体外和体内实验验证该系统对基因组DNA进行单碱基编辑的可行性；进而以几丁质合成酶基因（EcCHS）作靶标，利用该系统对编码EcCHS典型Chitin_synth_2结构域中重要氨基酸位点进行单（多）碱基替换，阐释EcCHS在脊尾白虾繁殖发育、生长与蜕皮等生理生化过程中的作用，进一步验证EcCDA与EcADA作为单碱基编辑工具酶的可行性。本项目的顺利实施，有助于推动脊尾白虾功能基因组的研究。

对于基因组中存在大量SNP位点的海洋无脊椎动物而言，单碱基基因编辑在开展海洋无脊椎动物SNP位点的功能研究方面具有其他基因编辑方式不可比拟的优势。开展甲壳动物单碱基基因编辑有助于揭示甲壳动物功能基因SNP位点（尤其是一些引起功能基因异义突变的SNP位点）与甲壳动物繁殖发育、生长、抗逆之间的关系，进而阐明甲壳动物的生命活动规律。本项目以脊尾白虾为研究对象，获得了脊尾白虾胞苷脱氨酶EcCDA和腺苷脱氨酶EcADA基因的全长cDNA序列；分别构建了EcCDA和EcADA与D10A缺陷型Cas9的融合表达重组质粒pnCas9-EcCDA和pnCas9-EcADA；利用体外转录的nCas9-EcCDA mRNA与体外转录NdMIH gRNA显微共注射中华米虾I细胞期受精卵，成功突变了与中华米虾生长发育相关重要功能基因NdMIH，并获得了多个位点发生单碱基突变（C→T）的突变体；利用体外转录的nCas9-EcADA mRNA与体外转录NdMIH gRNA显微共注射中华米虾I细胞期受精卵，成功突变了与中华锯齿米虾生长发育相关重要功能基因NdMIH，并获得了多个位点发生单碱基突变（A→G）的突变体；成功建立了脊尾白虾EcCDA和EcADA的单碱基编辑平台。利用CRISPR/Cas9技术分别敲除了脊尾白虾EcNinaB-X1和EcBCO2，并培育了其纯合突变体EcNinaB-X1-KO和EcBCO2-KO。并对纯合体突变体的体色变化与抗病力进行了测试，突变体与野生型相比，其肝胰脏颜色发生明显变化，并且对致病菌的抵抗力明显增强。实现了基于脊尾白虾卵黄蛋白结合蛋白VBP介导外源蛋白导入受精卵的非显微注射技术，为后期开展基于非显微注射的单碱基基因编辑提供了理论基础。获得了脊尾白虾几丁质合成酶EcCHS基因，确定了其基因结构，对EcCHS基因对应的Chitin_synth_2结构域部分进行了单碱基编辑，目前工作正在进行中。