CN108823248A - 一种利用CRISPR/Cas9编辑陆川猪CD163基因的方法 - Google Patents
一种利用CRISPR/Cas9编辑陆川猪CD163基因的方法 Download PDFInfo
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Abstract
本发明公开了一种利用CRISPR/Cas9编辑陆川猪CD163基因的方法。本发明采用在陆川猪基因组的CD163基因的外显子7上设计两条gRNA，分别构建至pX458和Px458R载体上，使CD163基因发生DNA片段的精确删除而破坏外显子7所编码的SRCR5半个结构域，从而获得抗PRRSV感染的CD163基因编辑猪。本发明的方法通过双荧光筛选能够更加有效地破坏CD163的SRCR5结构域。
Description
技术领域
本发明涉及生物技术领域，具体来说，涉及一种利用CRISPR/Cas9编辑陆川猪CD163基因的方法。
背景技术
猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的一种以妊娠母猪繁殖障碍和仔猪的呼吸道症状为主要特征的高度传染性疾病。CD163是一种位于细胞膜上的富含半胱氨酸(SRCR)结构域的PRRSV细胞受体，在PRRSV感染后病毒的脱壳与核酸释放过程中发挥重要的作用。
CD163共含有9个SRCR结构域，其中SRCR5在病毒感染过程中发挥主要的作用，有研究表明，SRCR5的缺失或破坏能抑制PRRSV的感染。同时有体外实验证明，SRCR5主要由CD163基因的外显子7负责编码。该发现为构建抗PRRSV的CD163基因编辑动物提供了依据，即通过基因编辑技术结合体细胞核移植的方法，制备出SRCR5被破坏的抗PRRSV的CD163基因编辑动物。
Cas9和gRNA是CRISPR/Cas9系统的基本成分，gRNA用于特异位点识别，Cas9用于切割靶位点DNA。与传统的基因组编辑技术相比，CRISPR/Cas9系统的构建更加简便、快速、廉价。研究发现，当gRNA的靶位点位于同一条染色体上时，利用Cas9和多条gRNA共转细胞，可以产生两条gRNA靶位点之间DNA片段的删除，DNA片段删除能更有效地敲除目的基因。
“陆川猪”短、宽、肥、圆。背腰宽广凹下，腹大常拖地、毛色呈一致性黑白花。历史悠久，品质优良，因原产与广西东南部的陆川县而得名，现主要分布于玉林、钦州、梧州等地。是中国八大地方优良猪种之一，具有繁殖力高、母性好、抗逆性强、肉嫩味鲜、体型紧凑、遗传力稳定等优点。
发明内容
本发明的目的是针对以上要解决的技术问题，提供一种能够有效敲除目的基因的方法，其利用CRISPR/Cas9编辑猪CD163基因。
为了解决上述问题，本发明提供了一种利用CRISPR/Cas9编辑陆川猪CD163基因的方法，其在陆川猪基因组的CD163基因的外显子7上设计两条gRNA，分别构建至pX458和pX458R载体上，使CD163基因实现DNA片段的精确删除而破坏外显子7所编码的SRCR5结构域，得到抗PRRSV感染的CD163基因编辑猪；其中，所述CD163基因的外显子7的核苷酸序列如SEQ ID NO.1所示。
根据本发明的方法，用于打靶所述CD163基因的外显子7的所述两条gRNA分别为核苷酸序列如SEQ ID NO.2所示的单链DNA分子以及核苷酸序列如SEQ ID NO.3所示的单链DNA分子。
根据本发明的方法，通过CRISPR/Cas9进行基因编辑对应的精确删除区域为如SEQID NO.1所示的核苷酸序列自5’末端第24-147位的核苷酸。
根据本发明的方法，通过CRISPR/Cas9进行基因编辑的步骤如下：
步骤1：将核苷酸序列如SEQ ID NO.2所示的gRNA-10构建到能表达Cas9蛋白和报告基因EGFP的pX458载体上，得到Px458-gRNA-10；将核苷酸序列如SEQ ID NO.3所示的gRNA-134构建到能表达Cas9蛋白和报告基因DsRed的pX458R载体上，得到Px458R-gRNA-134；将所述pX458-gRNA-10和所述pX458R-gRNA-134共转染猪的离体胎儿肾细胞，得到CD163基因编辑细胞群；
步骤2：用于扩增CD163基因外显子7编辑区域的引物对对所述CD163基因编辑细胞群进行PCR扩增，通过T-A克隆方法检测PCR扩增产物，计算克隆中含有编辑型CD163基因的克隆比例，即为该CRISPR/Cas9系统编辑效率。
在一个优选的方面，所述用于扩增CD163基因外显子7编辑区域的引物对包括核苷酸序列如SEQ ID NO.4所示的单链DNA分子以及核苷酸序列如SEQ ID NO.5所示的单链DNA分子。
在一个优选的方面，所述编辑型CD163基因为野生型CD163基因外显子7中123bpDNA片段缺失使SRCR5结构域被破坏所得到的基因型。
本发明还提供了一种利用CRISPR/Cas9编辑CD163基因的基因编辑动物的制备方法。
根据本发明提供的方法，将上述方法制备的含有编辑型CD163基因的胎儿肾细胞通过体细胞核移植获得CD163基因编辑动物。
本发明提供了一种研究CD163基因在PRRSV感染过程中的作用的方法。
根据本发明提供的方法，将上述方法制备的含有编辑型CD163基因的胎儿肾细胞通过体细胞核移植获得CD163基因编辑动物，制备出SRCR5被破坏的CD163基因编辑动物，从而研究CD163基因在PRRSV感染过程中的作用。
实验证明，本发明在目的基因猪(陆川猪)的CD163基因的外显子7上分别设计两条gRNA，构建至pX330载体，使CD163基因实现DNA片段的精确删除而丧失功能，得到CD163基因编辑猪。传统的抵御PPRSV感染的方法主要是接种PRRSV弱毒苗进行免疫防治，虽有一定成效，但PPRSV变异快，很不容易对型预防。本方法利用高效的CRISPR/Cas9系统以及核移植技术，可以快速制备出SRCR5被破坏的CD163基因编辑猪，通过进一步选育，即可得到抗PPRSV的CD163基因编辑猪。
附图说明
图1为CRISPR/Cas9编辑猪CD163基因示意图。
图2为猪胎儿肾细胞细胞中gRNA-10和gRNA-134的删除效率鉴定结果。
图3为根据本发明的CD163基因编辑陆川猪图片。
具体实施方式
下述实施例中所使用的试验方法如无特殊说明，均为常规方法。
所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
本发明所述的野生型CD163基因外显子7的核苷酸序列如序列表中的SEQ ID NO.1所示。
本发明所述的编辑型CD163基因为野生型CD163基因外显子7中124bp大片段缺失使SRCR5结构域被破坏所得到的基因型。通过CRISPR/Cas9进行基因编辑对应的精确删除区域为核苷酸序列如SEQ ID NO.1所示的自5’末端第24-147位的核苷酸。
针对CD163基因外显子7所设计两条gRNA，当将其分别构建到能表达Cas9蛋白的pX330载体上，就形成了两种能够特异性识别CD163基因并对识别位点进行打靶的CRISPR/Cas9系统。
这两种CRISPR/Cas9系统共转染细胞后，分别打靶CD163基因上相应gRNA所识别的位点，从而删除CD163基因上两条gRNA所识别位点的中间序列，实现猪CD163基因上外显子7的DNA片段的精确删除。
实施例1
利用CRISPR/Cas9编辑CD163基因制备基因编辑陆川猪
1、离体猪胎儿肾细胞的获得
将猪胎儿肾细胞从陆川猪胎儿肾脏中分离，在超净台内进行猪胎儿肾细胞的分离。用剪刀和镊子取下胎儿的肾脏组织，将取下的组织依次在75％酒精以及添加了抗生素的PBS里反复清洗，用小剪刀将组织块剪至1立方毫米大小，1600rpm离心5min去除PBS，再加入带抗生素的20％FBS的DMEM，轻轻吹打均匀，放入37℃细胞培养箱培养。放入细胞培养箱后，不要挪动培养皿，三天后，可观察到猪胎儿肾细胞已爬满至整个培养皿，再进行一般传代细胞的消化培养即可。
2、含有编辑型CD163基因的细胞的获得
1)质粒转染进细胞获得CD163基因编辑细胞
针对猪CD163基因外显子7设计两条gRNA，将其分别构建到能表达Cas9蛋白和荧光基因的pX458和pX458R载体上，形成两种能够特异性识别CD163基因并对识别位点进行打靶的CRISPR/Cas9系统(见图1)，即pX458-gRNA-10和pX458R-gRNA-134。
上述用于编辑猪CD163基因的两条gRNA序列如下：
gRNA-10：5’-ggaaacccaggctggttgga-3’(SEQ ID NO.2)；
gRNA-134：5’-ggaactacagtgcggcactg-3’(SEQ ID NO.3)。
采用电转的方法将5ug pX458-gRNA-10和5ug pX458R-gRNA-134共转染1×106猪胎儿肾细胞细胞，得到CD163基因编辑细胞。电转严格按照试剂盒和电转仪说明书操作。
2)鉴定含有编辑型CD163基因的细胞
设计用于扩增被删除区域的引物对如下：
CD163-DF3：5’-ctgctcagcccacaggaaac-3’(SEQ ID NO.4)；
CD163-DR3：5’-gccattcaccaagcggattt-3’(SEQ ID NO.5)。
将上述步骤1)得到的编辑细胞基因组DNA作为模板，用CD163-DF3与CD163-DR3组成的引物对进行PCR扩增。
如图2所示，分别回收约441bp(野生型条带大小)和317bp(删除目标区域后的条带大小)扩增产物并连接至T载体进行测序分析。计算克隆中含有编辑型CD163基因的克隆比例，即为该CRISPR/Cas9系统编辑效率，编辑效率越高，获得CD163基因编辑猪的比例越高。结果如以下表1所示。
表1
实施例2
利用体细胞核移植技术构建CD163基因编辑猪
1、体细胞核移植获得CD163基因编辑猪
从健康陆川母猪体内采取挑选发育阶段适宜的卵巢，用注射器抽取卵巢表面直径在3-5mm的卵泡中的内含物，将内含物在TL-PVA中稀释并重悬形成悬浊液。将悬浊液在37℃环境下静置至卵母细胞沉淀完全，将沉淀吸出置于在体视镜下用移液器或口吸管挑选卵周细胞完整的卵母细胞。将挑选的健康卵母细胞放入含有10％(重量百分比)卵泡液、FSH、LH、EGF的TCM-199中培养22h。再用移液器或口吸管将卵母细胞移到含有10％(重量百分比)卵泡液、EGF的TCM-199中继续培养22h。经过44h培养成熟后挑选已经排出第二极体的健康成熟卵母细胞作克隆胚胎用。
将上述制备的陆川猪的含有编辑型CD163基因的细胞，于5％(体积浓度)CO2、37℃饱和湿度的细胞培养箱培养，待细胞长至对数生长期时，即可用于核移植操作。
待卵母细胞体外培养成熟后，用采用电融合法将含有编辑型CD163基因的细胞群进行体细胞核移植，并在24h之内进行胚胎移植，制备不同品种的CD163基因型的基因编辑猪(见图3)。
2、CD163基因编辑猪的鉴定
采取少量CD163基因编辑猪的耳组织样提取基因组作为模板，用上述CD163-DF3与CD163-DR3组成的引物对(SEQ ID NO.4和SEQ ID NO.5)进行PCR扩增，并克隆测序，鉴定克隆猪的基因型，结果如以下表2所示。
表2
序列表
<110> 中山大学
<120> 一种利用CRISPR/Cas9编辑陆川猪CD163基因的方法
<160> 5
<170> SIPOSequenceListing 1.0
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cccacaggaa acccaggctg gttggagggg acattccctg ctctggtcgt gttgaagtac 60
aacatggaga cacgtggggc accgtctgtg attctgactt ctctctggag gcggccagcg 120
tgctgtgcag ggaactacag tgcggcactg tggtttccct cctgggggga gctcactttg 180
gagaaggaag tggacagatc tgggctgaag aattccagtg tgaggggcac gagtcccacc 240
tttcactctg cccagtagca ccccgccctg acgggacatg tagccacagc agggacgtcg 300
gcgtagtctg ctcaa 315
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ggaaacccag gctggttgga 20
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ggaactacag tgcggcactg 20
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ctgctcagcc cacaggaaac 20
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gccattcacc aagcggattt 20
Claims (6)
1.一种利用CRISPR/Cas9编辑陆川猪CD163基因的方法，其特征在于：在陆川猪基因组的CD163基因的外显子7上设计两条gRNA，分别构建至pX458和pX458R载体上，使CD163基因实现DNA片段的精确删除而破坏外显子7所编码的SRCR5结构域，得到抗PRRSV感染的CD163基因编辑猪；其中，所述CD163基因的外显子7的核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的方法，其特征在于：用于打靶所述CD163基因的外显子7的所述两条gRNA分别为核苷酸序列如SEQ ID NO.2所示的单链DNA分子以及核苷酸序列如SEQ IDNO.3所示的单链DNA分子。
3.根据权利要求1或2所述的方法，其特征在于：通过CRISPR/Cas9进行基因编辑对应的精确删除区域为如SEQ ID NO.1所示的核苷酸序列自5’末端第24-147位的核苷酸。
4.根据权利要求2或3中任一项所述的方法，其特征在于，通过CRISPR/Cas9进行基因编辑的步骤如下：
步骤1：将核苷酸序列如SEQ ID NO.2所示的gRNA-10构建到能表达Cas9蛋白和报告基因EGFP的pX458载体上，得到Px458-gRNA-10；将核苷酸序列如SEQ ID NO.3所示的gRNA-134构建到能表达Cas9蛋白和报告基因DsRed的pX458R载体上，得到Px458R-gRNA-134；将所述pX458-gRNA-10和所述pX458R-gRNA-134共转染猪的离体胎儿肾细胞，得到CD163基因编辑细胞群；
步骤2：用于扩增CD163基因外显子7编辑区域的引物对对所述CD163基因编辑细胞群进行PCR扩增，通过T-A克隆方法检测PCR扩增产物，计算克隆中含有编辑型CD163基因的克隆比例，即为该CRISPR/Cas9系统编辑效率。
5.根据权利要求4所述的方法，其特征在于：所述用于扩增CD163基因外显子7编辑区域的引物对包括核苷酸序列如SEQ ID NO.4所示的单链DNA分子以及核苷酸序列如SEQ IDNO.5所示的单链DNA分子。
6.根据权利要求4所述的方法，其特征在于：所述编辑型CD163基因为野生型CD163基因外显子7中123bp DNA片段缺失使SRCR5结构域被破坏所得到的基因型。
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| CN114774468B (zh) * | 2022-04-20 | 2022-12-20 | 温氏食品集团股份有限公司 | 一种等位基因分子标记及抗蓝耳病猪群体组建方法 |
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