实验海洋生物学重点实验室知识库

      CRISPR/Cas9 技术是第三代基因组编辑技术，主要用于基因功能的研究，遗传育种和疾病研究等。在鱼类中，模式鱼类和淡水鱼类应用较多，海水鱼类中应用较少。

  本研究探讨了 CRISPR/Cas9 技术在鱼类中最常见的两个应用：遗传育种和人类疾病研究。首先，以海水经济鱼类牙鲆（Paralichthys olivaceus）为研究对象，优化提升 CRISPR/Cas9 技术，推进该技术在海水鱼类遗传育种中的应用。其次，本研究以模式动物斑马鱼（Danio rerio）为研究对象，通过 CRISPR/Cas9 技术构建斑马鱼 mypn 突变体，研究肌节发育相关基因 mypn 与心肌病的关联，为心肌病的治疗提供新的科学依据。同时，在斑马鱼中尝试建立高通量突变体检测分析方法，为从大量牙鲆中筛选突变体提供借鉴。

      1、CRISPR/Cas9 基因组编辑技术在牙鲆中的优化提升

  目前海水鱼类基因组编辑技术尚未成熟，仍然存在突变率不稳定和成活率低的问题。牙鲆是我国重要海水经济养殖鱼类，由于牙鲆中基因组编辑技术尚未成熟且其繁殖周期长，获得纯合突变体时间长，因此优化提升基因组编辑技术，提高突变率和存活率，缩短获得牙鲆突变体的时间至关重要。在本文中，采用不同的 Cas9 mRNA 和 gRNA 注射浓度和纯化方式，通过对突变和孵化率分析来筛选合适的显微注射条件，并对纯化方式影响孵化率和畸形率的机理进行了初步分析。通过分析 nr3c1，fto，shisa2 基因组编辑后的突变率和孵化率，最终确定注射浓度为：Cas9 mRNA 500 ng/μL，gRNA 350 ng/μL。分析了两种常用的纯化方式——纯化柱纯化和 LiCl/乙醇沉淀纯化对胚胎的孵化率和畸形率的影响。结果显示与纯化柱纯化的 gRNA 相比，LiCl/乙醇沉淀纯化的 gRNA 在显微注射后牙鲆胚胎孵化率降低4.7%，畸形率增加19.6%。对原肠中期的胚胎进行转录组分析，结果表明 LiCl/乙醇沉淀的 gRNA 导致细胞凋亡相关通路和过程的基因显著上调，同时还影响了胚胎的神经系统发育和渗透压。

      2、斑马鱼 mypn 突变体的构建

      MYPN（肌钯蛋白）是横纹肌特异性蛋白，定位于肌节 Z 线、I 带和细胞核中，具有5个免疫球蛋白结构域。研究表明，在人体中 MYPN 的突变会引起扩张型心肌病、肥厚型心肌病、限制型心肌病、先天性肌无力等。本研究对斑马鱼 mypn 基因进行了克隆、氨基酸序列比对分析；通过 RT-PCR 和原位杂交分析 mypn 在不同胚胎时期的时空表达和成体组织中的表达；通过 CRISPR/Cas9 技术构建突变体，随后在个体水平和组织水平上对突变体胚胎和成鱼进行表型分析，并通过 RNA-seq 分析 mypn 缺失影响的信号通路。结果表明，斑马鱼 mypn 基因的 CDS 区存在一段串联重复序列，氨基酸多序列比对分析表明 MYPN 结构域高度保守，与人和小鼠的相似，斑马鱼 MYPN 也含有5个免疫球蛋白结构域。Mypn 为母源基因，在胚胎期的20 hpf 和24 hpf 时，表达量最高，20-36 hpf 时在体节中高表达，72 hpf 时在心脏中表达；其在成鱼的心脏、肌肉、眼睛和性腺中表达。Mypn 敲除后，共获得18种突变类型，最终选择 -302 bp 的突变体继续培育获得纯合突变体，进行心脏表型等分析。胚胎期，斑马鱼 mypn 纯合突变体的血液循环异常，心率显著下降，心脏形态和位置无明显变化。4月龄斑马鱼的心脏组织切片 HE 染色结果显示，纯合突变体和杂合突变体的心脏心室内腔面积占比显著缩小，心室壁显著增厚，表现为肥厚型心肌病的症状；天狼星红染色结果表明4月龄纯合突变体的心脏有间质纤维化的现象；透射电镜结果显示，4月龄纯合突变体心脏的心肌肌丝松散，出现肌丝局部缺失。2月龄斑马鱼心脏转录组数据分析显示纯合突变体中纤维化相关通路——TGF-β 信号通路上调，但是纤维化标志基因 tgfb1 和 ccn2a 的表达无明显差异，呈现下调趋势；纯合突变体和杂合突变体的肌动蛋白细胞骨架的调节通路均发生变化，但纯合突变体受到的影响更大；纯合突变体的心肌收缩通路下调，与胚胎期心率显著下降相对应，而杂合突变体心肌收缩通路却上调；纯合突变体和杂合突变体的心肌细胞肾上腺素传导通路也发生改变，其在纯合突变体中下调，而在杂合突变体中上调，分别与心肌收缩通路的变化一致。

      3、斑马鱼突变体筛选对牙鲆的指导

  本研究利用 STR 分型检测的方法对斑马鱼 mypn 突变体进行筛选，成功获得突变体，进而建立了突变体检测方法；在牙鲆中利用该方法实现了突变体的高通量筛选，节省了大量时间和成本。

      CRISPR/Cas9 is the third generation of genome editing technology, which is mainly used for gene function research, genetic breeding and disease research. Among fish, model fish and freshwater fish are widely used, while marine fish are less used.

      This study explored the two most common applications of CRISPR/Cas9 technology in fish: genetic breeding and human disease research. At first, the marine economic fish olive flounder (Paralichthys olivaceus) was used as the research object to optimize the CRISPR/Cas9 technology and promote the application of CRISPR/Cas9 technology in the genetic breeding of marine fish. Second, the model animal zebrafish (Danio rerio) was used as the research object. Through construction of mypn mutant using CRISPR/Cas9 technology, the relationship between mypn and cardiomyopathy was studied, which provided a new scientific basis for the treatment of cardiomyopathy. At the same time, high throughput mutant screening detection and analysis method were attempted to establish in zebrafish, which could provide base and reference for screening mutant from a large number of flounder.

      1. The optimization of CRISPR/Cas9 genome editing technology in olive  flounder

      The CRISPR/Cas9 genome editing technology of marine fish is not yet mature, and there are still problems of unstable mutation rate and low survival rate. Olive flounder is an important marine economic fish in China. It needs long time to obtain homozygous mutants due to the immature genome editing technology and long breeding cycle in olive flounder. Therefore, it is very important to optimize the genome editing technology, improve the mutation rate and survival rate, and shorten the time to obtain olive flounder mutants. Herein, different injection concentrations and purification methods were used for Cas9 mRNA and gRNA to screen suitable microinjection conditions by analyzing mutation rate and hatching rate. The mechanism of purification methods affecting hatching rate and deformity rate was preliminarily analyzed too. By analyzing the mutation rate and hatching rate after genome editing with nr3c1, fto, shisa2, the injection concentration was finally determined: Cas9 mRNA 500 ng/μL, gRNA 350 ng/μL. The effects of two common purification methods, column purification and LiCl/ethanol precipitation purification, on the hatching rate and deformity rate of embryos were analyzed. The results showed the hatching rate of the gRNA purified by LiCl/ethanol precipitation decreased by 4.7% and the deformity rate increased by 19.6% after microinjection comparison with that of the column purified. RNA-seq analysis of the mid-gastrula embryos showed that the apoptosis-related pathways and processes were significantly up-regulated after microinjection of LiCl/ethanol-precipitated gRNA, and the development of embryonic nervous system and osmotic pressure were affected, too.

      2. Construction of zebrafish mypn mutant

      MYPN (myopalladin) is a striated muscle-specific protein, which is located in the Z-line, I-band and nucleus of the sarcomere and has five immunoglobulin domains. Studies have shown that MYPN mutations in the human body can cause dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, congenital myasthenia and so on. In this study, the mypn gene was cloned from zebrafish and its deduced amino acid sequence was alignment. The temporal and spatial expression of mypn in different embryonic stages and adult tissues were analyzed by RT-PCR and in situ hybridization. The mutants were constructed by CRISPR/Cas9 technology, and then the phenotypes of mutant embryos and adult fish were analyzed at the individual level and tissue level, and the signaling pathways affected by mypn deletion were analyzed by RNA-seq. The results showed that there was a tandem repeat sequence in the CDS region of zebrafish mypn gene. Amino acid multiple sequence alignment showed that the MYPN domain was highly conserved and similar to human and mouse. Zebrafish MYPN also had five immunoglobulin domains. Mypn is a maternal gene, and the expression of mypn is the highest at 20 hpf and 24 hpf in the embryonic period.It is highly expressed in the somite from 20-36 hpf, and expressed in the heart at 72 hpf. It is expressed in the heart, muscle, eyes and gonads of adult fish. A total of 18 mutation types were obtained during mypn knockout. Finally, the mutant of -302 bp was selected to continue to culture to obtain homozygous mutants for future analysis such as heart phenotype. The homozygous mutant of zebrafish mypn showed abnormal blood circulation, decreased heart rate during embryonic period, and there was no significant change in the shape and location of the heart. The results of HE staining on heart tissue sections of 4-month-old zebrafish showed that the proportion of ventricular cavity area was significantly reduced in the heart of homozygous mutant and heterozygous mutant and the ventricular wall was significantly thickened, presenting as the symptoms of hypertrophic cardiomyopathy. Sirius red staining results showed that the heart of homozygous mutant had fibrosis at 4 months of age. The results of transmission electron microscopy showed that the myocardial filaments of 4-month-old zebrafish were loose with partially missed. RNA-seq analyzed showed that the fibrosis-related pathway—TGF-β signaling pathway was up-regulated in the heart of 2-month-old zebrafish homozygous mutant, but the expression of fibrosis marker genes tgfb1 and ccn2a was downregulated with not significantly different. The regulation of actin cytoskeleton was both affected in homozygous mutants and heterozygous mutants, and it was serious in the homozygous mutants. The myocardial contraction pathway was down-regulated in the heart of homozygous mutant, which was consistent with a significant decrease in embryonic heart rate, while the myocardial contraction of heterozygous mutant was up-regulated. The adrenaline conduction pathway of cardiomyocytes was affected in both of homozygous mutants and heterozygous mutant. It was was down-regulated in the heart of homozygous mutant, while it was up-regulated in the heterozygous mutant, which was consistent with the changes of myocardial contraction pathway, respectively.

      3. The guidance of zebrafish mutant screening method for that of olive flounder

      In this study, STR typing was successfully used to screen zebrafish mypn mutants. Mutants were got. Then, the mutant screening method using STR was established. High-throughput screening of mutants was achieved in olive flounder using this method, which saved a lot of time and cost.

第1章 绪论      1

1.1  基因组编辑技术 1

1.1.1  CRISPR/Cas9 技术 1

1.1.2  鱼类中的基因组编辑     2

1.2  海水鱼类基因组编辑遗传育种存在的问题及影响因素 3

1.3  心脏发育及结构 6

1.3.1  斑马鱼心脏发育     6

1.3.2  心肌结构 7

1.4  心肌病 9

1.4.1  扩张型心肌病 9

1.4.2  肥厚性心肌病 11

1.5  MYPN 13

1.5.1  MYPN 蛋白的结构与功能    13

1.5.2  MYPN 的研究进展 14

1.6  研究目的及意义 14

第2章 CRISPR/Cas9 技术在牙鲆中的优化   17

2.1  研究背景    17

2.2  材料与方法 17

2.3  结果    26

2.3.1  Cas9 mRNA 和 gRNA 注射浓度对牙鲆基因组编辑的影响       26

2.3.2  Cas9 mRNA 和 gRNA 纯化方式对牙鲆基因组编辑的影响       28

2.4  讨论    35

2.4.1  Cas9 mRNA 和 gRNA 注射浓度对牙鲆基因组编辑的影响       35

2.4.2  Cas9 mRNA 和 gRNA 纯化方式对牙鲆基因组编辑的影响       36

2.5  小结    38

第3章 CRISPR/Cas9 技术在斑马鱼 mypn 心肌病模型中的应用       39

3.1  研究背景    39

3.2  材料与方法 39

3.2.1  实验鱼与样品采集 39

3.2.2  实验试剂与仪器     40

3.2.3  血红蛋白染色 41

3.2.4  总 RNA 提取和 cDNA 合成       41

3.2.5  斑马鱼 mypn 基因克隆、验证   41

3.2.6  半定量 PCR    42

3.2.7  原位杂交 43

3.2.8  生物学信息分析     46

3.2.9  斑马鱼 mypn 基因敲除       46

3.2.10  斑马鱼 mypn 纯合突变体的获得     48

3.2.11  斑马鱼心脏组织切片   49

3.2.12  超薄切片      49

3.2.13  鬼笔环肽染色      50

3.2.14  天狼星红染色      50

3.2.15  RNA-seq      50

3.2.16  荧光实时定量 PCR（qPCR）验证    50

3.3  结果    52

3.3.1  斑马鱼胚胎心脏发育与血液循环 52

3.3.2  斑马鱼 mypn 基因克隆与序列分析   54

3.3.3  斑马鱼 MYPN 蛋白结构分析      60

3.3.4  斑马鱼 mypn 基因在胚胎期的时空表达和成体组织表达       60

3.3.5  斑马鱼 mypn 基因的敲除   63

3.3.6  斑马鱼 mypn 纯合突变体的获得       65

3.3.7  斑马鱼 mypn 突变体胚胎期表型       69

3.3.8  斑马鱼 mypn 突变体心脏表型   71

3.3.9  斑马鱼心脏转录组分析 74

3.4  讨论    84

3.4.1  斑马鱼胚胎心脏发育与血液循环情况 84

3.4.2  斑马鱼mypn 基因序列分析及蛋白结构    84

3.4.3  斑马鱼mypn 基因的表达模式    85

3.4.4  斑马鱼mypn 纯合突变体的获得及表型分析    85

3.5  小结    87

第4章 斑马鱼突变体的筛选对牙鲆突变体筛选的指导      89

4.1  研究背景    89

4.2  材料与方法 89

4.2.1  实验鱼与样品采集 89

4.2.2  实验试剂与仪器     89

4.2.3  STR 分型检测 89

4.3  结果    90

4.3.1  STR 分型技术在突变体筛选中的建立及应用    90

4.3.2  F1 代纯合突变体的获得      91

4.4  讨论    92

4.5  小结    92

第5章 结论与展望   93

参考文献     95

致谢     105

作者简历及攻读学位期间发表的学术论文与其他相关学术成果 107