阿勒泰羊胚胎PDGFD基因编辑研究

doi:10.11843/j.issn.0366-6964.2025.04.019

摘要/Abstract

摘要：

旨在用CRISPR/Cas9技术生产阿勒泰羊血小板源生长因子D(PDGFD)基因SNP位点的基因编辑胚胎。本研究根据阿勒泰羊PDGFD基因脂尾性状相关的3个SNPs位点设计合成5个sgRNA (PDGFD-314-sgRNA2、PDGFD-314-sgRNA5、PDGFD-410-sgRNA、PDGFD-397-sgRNA2、PDGFD-397-sgRNA7)，结合Cas9核酸酶进行体外活性分析。电转阿勒泰羊体外受精4~5 h的胚胎Cas9-sgRNA核糖核蛋白复合物(RNP)，每组35~40枚，3次重复，培养至第7天统计囊胚率，进行基因组靶基因位点扩增及测序分析。受精卵电转PDGFD和MSTN基因(非PDGFD基因)同源重组基因编辑材料，培养至第7天统计囊胚率及进行PDGFD sgRNA脱靶效率分析。结果表明，5个sgRNA在体外均有高效的切割活性；电转受精卵的存活率和卵裂率与对照组差异均不显著(P>0.05)，但电转组囊胚率显著低于对照组(P < 0.05)。PDGFD-397-sgRNA2在绵羊体外胚胎中的编辑效率均高于其它sgRNAs。PDGFD-397-sgRNA2在绵羊基因组上错配1个碱基的脱靶位点有19个，覆盖在外显子区的基因有CFDP1，APOO，PCDH11X等；错配2个碱基的位点有31个，覆盖在外显子区的基因有TIMM17B、C1H1orf228和TLL1等。电转PDGFD和MSTN基因同源重组编辑材料，两组胚胎的存活率和卵裂率差异不显著(P>0.05)，但前者的囊胚率显著低于后者(P < 0.0 5)。本研究结果表明，阿勒泰羊PDGFD基因尾脂性状相关SNP位点的CRISPR/Cas9编辑显著降低体外胚胎的囊胚率，因此，PDGFD基因可能影响绵羊早期胚胎的发育。综上所述，PDGFD基因可能对绵羊胚胎的早期发育有着重要的作用。

关键词: 电转, CRISPR/Cas9, PDGFD, 胚胎发育

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 nuclease 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 cleavage 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 electroperforated 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 significantly 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 significantly 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.

Key words: electric transmission, CRISPR/Cas9, PDGFD, embryonic development

中图分类号:

S826.2

马秀玲, 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, 吴阳升. 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.

MA Xiuling, ZHANG Xinru, CHEN Ying, LIANG Hongyan, ABDUREYIMU Gulimire, WANG Liqin, LIN Jiapeng, LI Weijian, WANG Xuguang, WU Yangsheng. PDGFD Gene Editing in Altay Sheep Embryos[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1700-1711.

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序号 Number	sgRNA名称 Name of sgRNA	核心序列 Core sequence	基因位置 Location of genes
1	PDGFD-314-sgRNA2	GGACTAATATCAGTGAAACG	Oar_v3.1，Chr15:3859314
2	PDGFD-314-sgRNA5	CTAATATCAGTGAAACGGGG	Oar_v3.1，Chr15:3859314
3	PDGFD-410-sgRNA	GACGTACCACTCTGAGCACA	Oar_v.4.0, Chr15:3904410
4	PDGFD-397-sgRNA2	TCAGTGCCTGGCTCAAATAG	Oar_Rambouillet v1.0, Chr15:4172397
5	PDGFD-397-sgRNA7	TGGCTCAAATAGCGGCTTGT	Oar_Rambouillet v1.0, Chr15:4172397

序号 Number	引物名称 Primer name	引物序列 Primer sequence	产物/bp Product
1	PDGFD-410-F1	TCTGTTGTCGCTTAGAGTTT	616
2	PDGFD-410-F2	AAAAGTGAAAGGGAAGTCG	415
3	PDGFD-410-R	TAGAAGGATGGCAGAAAGG
4	PDGFD-314-F1	AAAATGAGTGGTATGTGGGTG	597
5	PDGFD-314-F2	GGGCTAAGGTCCCTGGAGT	409
6	PDGFD-314-R	GGGAAATTCTTGTGGCAGT
7	PDGFD-397-F	GATTGCCCTCCAGATACCA
8	PDGFD-397-R1	GAGTGGTCTAAGCGGTTTT	746
9	PDGFD-397-R2	CTGCTTCATTCCACCTTCC	474
10	PDGFD-397-HIT-F	ggagtgagtacggtgtgcGATTGCCCTCCAGATACCA	262
11	PDGFD-397-HIT-R	gagttggatgctggatggGGACTGTCCCTCTTTGGGT

位点 Site	PDGFD SNP位点[文献] SNP loci in PDGFD	哈萨克羊对应位置及碱基类型 Kazakh sheep corresponding position and base type
1	Oar_v3.1：Chr15:3622093(C>T)^[16]	3 817 380 (T)
2	Oar_v3.1：Chr15:3859314(T>C)^[15]	4 066 876 (C)
3	Oar_v3.1：Chr15:4031794(A>C)^[17]	4 242 267 (G)
4	Oar_v.4.0：Chr15:3852134(C>T)^[18]	4 055 473 (C)
5	Oar_v.4.0：Chr15:3904410(G>A)^[14]	4 107 625 (A)
6	Oar_v.4.0：Chr15:4122606(C>G)^[18]	4 326 797 (G)
7	Oar_Rambouillet v1.0：Chr15:4172397(A>G)^[19]	3 827 484 (G)

编辑类型 Editing type	总卵数/枚 Total number of oocytes	死亡率/% Mortality rate	24 h卵裂率/% 24 h cleavage rate	48 h卵裂率/% 48 h cleavage rate	7 d囊胚率/% Blastocyst rate on day 7
PDGFD-314-sgRNA2	109	11.96±0.04	46.42±0.12	90.37±0.09	35.70±0.02^B
PDGFD-314-sgRNA5	107	12.22±0.09	42.24±0.04	89.86±0.10	39.92±0.05^b
PDGFD-410-sgRNA	109	16.98±0.09	44.00±0.12	93.75±0.08	36.77±0.01^B
PDGFD-397-sgRNA2	112	19.03±0.09	42.60±0.13	90.33±0.13	32.12±0.10^B
PDGFD-397-sgRNA7	115	10.95±0.06	45.55±0.12	90.69±0.03	36.20±0.01^B
对照组Control	118	1.60±0.03	49.89±0.02	79.34±0.08	57.73±0.05^Aa

编辑类型 Editing type	总卵数/枚 Total number of oocytes	死亡率/% Mortality rate	24 h卵裂率/% 24 h cleavage rate	48 h卵裂率/% 48 h cleavage rate	7 d囊胚率/% Blastocyst rate on day 7
PDGFD-397-sgRNA2电转编辑组 PDGFD-397-sgRNA2 electrotransfer editing group	131.00	7.36±0.08	54.31±0.91	94.26±0.06	29.68±0.00^b
MSTN电转编辑组 MSTN electrotransfer editing group	120.00	11.79±0.06	60.67±0.14	92.81±0.02	51.99±0.15^a