阿勒泰羊胚胎PDGFD基因编辑研究

doi:10.11843/j.issn.0366-6964.2025.04.019

Abstract

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

CLC Number:

S826.2

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.

Figures/Tables 10

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References 40

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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

PDGFD Gene Editing in Altay Sheep Embryos

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错配碱基数 Mismatched base numbers	sgRNA类型sgRNA type
错配碱基数 Mismatched base numbers	PDGFD314-sgRNA2	PDGFD314-sgRNA5	PDGFD410-sgRNA	PDGFD397-sgRNA2	PDGFD397-sgRNA7
0	0	0	0	1	0
1	1	1	0	19	1
2	0	0	2	31	2
合计Total	1	1	2	51	3

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