猪CD163基因的单碱基编辑研究

The Study of Base Editing of Porcine CD163 Gene

旨在利用YE1-BE3-FNLS载体对猪的CD163基因进行C>T的单碱基突变,形成终止密码子TAA,从而导致CD163基因翻译的提前终止。利用网站在猪CD163基因的第7外显子筛选到高效率的sgRNA序列使其符合C5位点,并且C5后续的两个碱基为AA,CAA与起始密码子ATG之间的碱基数为3的整数倍。PCR扩增CD163-sgRNA和YE1-BE3-FNLS载体中的APOBEC-Cas9n-UGI片段,通过体外转录形成CD163-sgRNA和APOBEC-Cas9n-UGI mRNA。体外培养猪卵母细胞并进行孤雌激活,利用显微操作仪对孤雌胚胎进行CD163-sgRNA和APOBEC-Cas9n-UGI mRNA注射,待胚胎发育至桑椹胚和囊胚阶段,提取胚胎基因组DNA对单碱基编辑区域进行PCR扩增测序。结果显示,在49个候选sgRNA中成功筛选出一个CD163-sgRNA,利用PCR成功扩增了CD163-sgRNA和APOBEC-Cas9n-UGI片段,注射CD163-sgRNA和APOBEC-Cas9n-UGI mRNA后的猪孤雌胚胎发育到2~4细胞期。挑选注射的20个2~4细胞期胚胎经过PCR扩增与产物测序后发现有12个胚胎的CD163基因的第7外显子的预期位点C(num1479)>T,单碱基编辑效率约60%。对CD163-sgRNA的off-target分析发现,在错配3个碱基的情况下该CD163-sgRNA有5个off-target区域,对这5个区域进行PCR扩增和sanger测序分析后发现都没有发生C>T的突变。本研究利用YE1-BE3-FNLS工具在胚胎水平上对猪的CD163基因进行了单碱基编辑,成功使得该基因第7外显子预期位点(num1479)C变为T,从而使得密码子CAA变为TAA,可提前终止CD163基因的翻译。

The aim of this study was to use the YE1-BE3-FNLS vector to carry out a single-base mutation of C>T in the porcine CD163 gene to form the stop codon TAA,which would lead to the premature termination of the translation of the CD163 gene.The website was used to design high-efficiency sgRNA sequence at the 7th exon of porcine CD163 gene and make it conform to C5 feature,and the following two bases of C5 were AA,and the number of bases between CAA and the start codon ATG was the integer multiple of 3.The CD163-sgRNA and APOBEC-Cas9n-UGI fragment in the YE1-BE3-FNLS vector was amplified by PCR,and CD163-sgRNA and APOBEC-Cas9n-UGI mRNA were produced by in vitro transcription.Porcine oocytes were cultured in vitro and undergone parthenogenetic activation.The CD163-sgRNA and APOBEC-Cas9n-UGI mRNA were injected into parthenogenetic embryos by micromanipulator.After the embryo developed to the stage of morula and blastocyst,the genomic DNA of the embryo was extracted and its single-base editing region was subjected to PCR amplification and sequencing.The results showed that a CD163-sgRNA was successfully screened among 49 candidate sgRNAs.The CD163-sgRNA and APOBEC-Cas9n-UGI fragments were successfully amplified by PCR,and the porcine parthenogenetic embryos developed to the 2-4 cell stage after injection of CD163-sgRNA and APOBEC-Cas9n-UGI mRNA.After PCR amplification and product sequencing of selected injected 20 embryos at 2-4 cell stage,there were 12 embryos with the expected site C(num1479)>T of the 7th exon of the CD163 gene,and the single-base editing efficiency was about 60%.It was found that the CD163-sgRNA had 5 off-target regions in the case of a mismatch of 3 bases based on the off-target analysis.After analyzing these 5 regions,it was found that there was no C>T mutation using PCR amplification and sanger sequencing.The pig CD163 gene was suffered with base editing by YE1-BE3-FNLS tool at the embryo level in this study,and the 7th exon expected site(num1479)C was successfully changed to T,which can terminate the translation of CD163 gene in advance by making the codon CAA to TAA.