畜牧兽医学报2025，Vol.56Issue(4)：1700-1711,12.DOI:10.11843/j.issn.0366-6964.2025.04.019
阿勒泰羊胚胎PDGFD基因编辑研究
PDGFD Gene Editing in Altay Sheep Embryos
摘要
Abstract
The study aimed to generate gene edited embryos of single nucleotide polymorphisms(SNPs)of platelet-derived growth factor D(PDGFD)gene in Altay sheep using CRISPR/Cas9 technology.Five sgRNAs(PDGFD-314-sgRNA2,PDGFD-314-sgRNA5,PDGFD-410-sgRNA,PDGFD-397-sgRNA2,PDGFD-397-sgRNA7)were designed and synthesized based on 3 SNP loci related to lipid tail traits of PDGFD gene in Altay sheep,combined with Cas9 nucle-ase for in vitro activity analysis.Electroporation of Cas9-sgRNA ribonucleoprotein(RNP)in Altay sheep embryos 4-5 h after in vitro fertilization,35-40 embryos per group,triple repetition.The blastocyst rate was counted on the 7th day of in vitro culture,and the genomic target gene loci were amplified and sequenced.PDGFD and MSTN genes(non-PDGFD gene)homologous recombination gene editing materials were electroporated into zygotes,and the blastocyst rate and PDGFD sgRNA off-target efficiency were analyzed on the 7th day of culture.All 5 sgRNAs had high cleavage activity in vitro.There was no significant difference in the survival rate and cleav-age rate between the electroporated zygotes and control groups(P＞0.05),but the blastocyst rate in the electroporation group was significantly lower than that in the control group(P＜0.05).The editing efficiency of PDGFD-397-sgRNA2 was higher than other sgRNAs in embryos.There were 19 off-target sites with one-base mismatch of PDGFD-397-sgRNA2 in the sheep genome,and the genes covering the exon region included CFDP1,APOO,PCDH11X,etc.There were 31 2-base mismatch sites,and TIMM17B,C1H1orf228 and TLL1 genes covered the exon region.PDGFD and MSTN genes homologous recombination editing materials were elec-troperforated respectively,there was no significant difference in the survival rate and cleavage rate between the two groups(P＞0.05),but the blastocyst rate of the PDGFD group was sig-nificantly lower than that of the MSTN group(P＜0.05).The results of this study showed that CRISPR/Cas9 editing of SNP locus related to tail fat trait of PDGFD gene in Altay sheep signifi-cantly reduced the blastocyst rate of in vitro embryos,suggesting that PDGFD gene may affect the early embryo development of sheep.In conclusion,PDGFD gene may play an important role in the early development of ovine embryo.关键词
电转/CRISPR/Cas9/PDGFD/胚胎发育Key words
electric transmission/CRISPR/Cas9/PDGFD/embryonic development分类
农业科技引用本文复制引用
马秀玲,吴阳升,张欣如,陈莹,梁红艳,古丽米热·阿布都热依木,汪立芹,林嘉鹏,李伟健,王旭光..阿勒泰羊胚胎PDGFD基因编辑研究[J].畜牧兽医学报,2025,56(4):1700-1711,12.基金项目
新疆"三农"骨干人才培养项目(2022SNGGGCC036) （2022SNGGGCC036）
新疆维吾尔自治区重大科技专项项目(2023A02011-2) （2023A02011-2）
新疆维吾尔自治区羊产业技术体系(XJARS-09-08) （XJARS-09-08）
科技创新2030-农业生物育种重大项目(2022ZD0401303) （2022ZD0401303）
