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CN-122012627-A - Porcine MSTN, pAPN and CD163 gene synchronous editing embryo and preparation method and application thereof

CN122012627ACN 122012627 ACN122012627 ACN 122012627ACN-122012627-A

Abstract

The invention relates to the field of animal genetic engineering and biotechnology, in particular to a pig MSTN, pAPN and CD163 gene synchronous editing embryo, a preparation method and application thereof. Based on CRISPR/Cas9 technology, MSTN exon 1, CD163 exon 7 and pAPN exon 2 are selected as target areas, high-efficiency sgRNA is designed and screened, integrated tandem editing vectors containing three-gene sgRNA expression cassettes are constructed through homologous recombination, positive PEF cells are obtained through puromycin screening and sequencing identification after electrically transfected pig embryo fibroblasts, and then synchronous editing embryos are prepared through somatic cell nuclear transfer. The three-gene synchronous editing efficiency of the invention reaches 8.9%, the embryo fusion rate is more than or equal to 70%, the cleavage rate is more than or equal to 75%, the blastula rate is more than or equal to 12%, the development capability is normal, the synchronous improvement of the growth performance and disease resistance related genes is realized, and the invention has important industrialized application value.

Inventors

  • TANG ZHONGLIN
  • CHEN ZHILONG
  • XIE HAO
  • PENG CUITING
  • PAN YINGTING
  • QI LIN
  • ZHAO YULAN

Assignees

  • 中国农业科学院深圳农业基因组研究所(岭南现代农业科学与技术广东省实验室深圳分中心)

Dates

Publication Date
20260512
Application Date
20260212

Claims (10)

  1. 1. The preparation method of the porcine MSTN, pAPN and CD163 gene synchronous editing embryo is characterized by comprising the following steps: (1) Selecting target areas aiming at functional domains of pig MSTN, pAPN and CD163 genes, designing sgRNA and constructing a single-gene sgRNA expression vector, and screening sgRNA with high editing efficiency of each gene after electrically transfecting pig embryo fibroblasts; (2) Assembling the sgRNA expression cassette obtained by screening in the step (1) into an integrated tandem expression system by utilizing a homologous recombination method, converting the integrated tandem expression system into competent cells, and obtaining a three-gene knockout vector through sequencing and identification; (3) Electrotransfection of the three-gene knockout vector into PEF cells, and drug screening and sequencing identification are carried out to obtain MSTN, pAPN, CD163 gene synchronously edited positive PEF cells; (4) And constructing a recombinant embryo by taking the positive PEF cells as donor cells and adopting a somatic cell nuclear transfer technology, and obtaining the porcine MSTN, pAPN and CD163 gene synchronous editing embryo through in-vitro culture and sequencing verification.
  2. 2. The method of claim 1, wherein in step (1), the targeting regions are MSTN exon 1, CD163 exon 7, and pAPN exon 2.
  3. 3. The method of claim 1, wherein in step (1), 2 pairs of sgrnas are designed for each targeting region, the sgrnas are linked to pX330 vector to construct a single-gene sgRNA expression vector, and the editing efficiency of the sgrnas 1 is higher than that of the sgrnas 2; The forward and reverse primers of the sgRNA1 sequence of the CD163 are shown as SEQ ID NO. 1-2; the forward and reverse primers of the sgRNA2 sequence of the CD163 are shown in SEQ ID NO. 3-4; The forward and reverse primers of the sgRNA1 sequence of the MSTN are shown as SEQ ID NO. 5-6; The forward and reverse primers of the sgRNA2 sequence of the MSTN are shown as SEQ ID NO. 7-8; the forward and reverse primers of the sgRNA1 sequence of pAPN are shown as SEQ ID NO. 9-10; The forward and reverse primers of the sgRNA2 sequence of pAPN are shown in SEQ ID NO. 11-12.
  4. 4. The method of claim 1, wherein in step (2) the integrated tandem expression system is a CRISPR/Cas9 editing vector constructed by homologous recombination comprising three pairs of sgRNA expression cassettes and Cas9 expression elements, and the competent cells are Fast-T1 competent cells.
  5. 5. The method according to claim 1, wherein in step (3), the drug is screened using puromycin at a concentration of 2.5 μg seed mL -1 , and positive PEF cells are identified by PCR amplification and Sanger sequencing after screening.
  6. 6. The method according to claim 1, wherein in the step (3), the mutation rate of the MSTN gene is 62%, the frame shift/non-frame shift ratio is 14:14, the mutation rate of the pAPN gene is 26%, the frame shift/non-frame shift ratio is 7:5, the mutation rate of the CD163 gene is 11%, the frame shift/non-frame shift ratio is 4:1, and the ratio of simultaneous editing of the three genes is 8.9%.
  7. 7. The method according to claim 1, wherein in step (4), the recombinant embryo is cultured in vitro, the cell fusion rate is not less than 70%, the embryo cleavage rate is not less than 75%, the embryo blastocyst rate is not less than 12%, and the gene editing result of embryo at blastocyst stage is consistent with positive PEF cells.
  8. 8. A synchronously edited pig MSTN, pAPN and CD163 genes embryo, wherein the embryo is obtained by the preparation method of any one of claims 1-7, and the MSTN gene, CD163 gene and pAPN gene in the embryo are mutated in their corresponding targeting regions, respectively.
  9. 9. Use of the porcine MSTN, pAPN and CD163 gene simultaneous editing embryos of claim 8 for breeding polygene editing pigs.
  10. 10. The use according to claim 9, wherein the resulting polygene editing pigs are bred for high lean meat percentage and disease resistance, wherein the MSTN gene knockout mediates lean meat percentage elevation, the pAPN gene editing reduces the risk of transmissible gastroenteritis virus infection, and the CD163 gene editing blocks the porcine reproductive and respiratory syndrome virus infection pathway.

Description

Porcine MSTN, pAPN and CD163 gene synchronous editing embryo and preparation method and application thereof Technical Field The invention relates to the field of animal genetic engineering and biotechnology, in particular to a pig MSTN, pAPN and CD163 gene synchronous editing embryo, a preparation method and application thereof. Background Pig is one of the core livestock varieties in animal husbandry, and the production performance (such as growth speed and lean meat percentage) and disease resistance of the pig are directly related to the economic benefit and the product quality safety of the breeding industry, so that the pig has important strategic significance for guaranteeing the sustainable development of animal husbandry. Along with the development of molecular biology and genetic engineering technology, gene editing has become a key means for pig genetic improvement, wherein the CRISPR/Cas9 technology is widely applied to pig character improvement research by virtue of high efficiency, accuracy and simple operation. The Myostatin (MSTN) gene is a key negative control factor for controlling animal muscle development, the function deficiency can obviously relieve the inhibiting effect on muscle growth, the lean meat percentage and the growth speed of pigs are greatly improved, and the gene is a core target gene for improving the production performance of the pigs. The aminopeptidase N (pAPN) gene is taken as an important receptor for infecting host cells by various viruses (such as transmissible gastroenteritis viruses of pigs and the like), the editing and transformation can reduce the susceptibility of pigs to related viruses, the CD163 gene is a key mediating molecule of the invasion cells of Porcine Reproductive and Respiratory Syndrome Viruses (PRRSV), and the knockout of the gene is proved to be an effective strategy for cultivating PRRSV-resistant pig varieties. Therefore, the synchronous improvement of MSTN mediated growth performance and pAPN and CD163 mediated disease resistance is a core target for cultivating high-quality disease-resistant pig new varieties. However, there are still many bottlenecks to be solved in the existing pig gene editing technology and related patent schemes: the single gene editing limitation is remarkable, the existing research is focused on editing single genes of MSTN, pAPN or CD163, only one property of pigs can be improved, the requirement of the breeding industry on synchronous improvement of 'high yield + disease resistance' multi-optimal properties cannot be met, and the double pressures of production efficiency and epidemic disease prevention and control in live pig breeding are difficult to fundamentally solve. The multi-gene editing efficiency is low and the compatibility is poor, the traditional multi-gene editing mostly adopts a multi-carrier cotransfection strategy, and the problems of mutual interference among carriers, scattered editing efficiency, high positive cell screening difficulty and the like exist, so that the success rate of synchronously editing a plurality of genes is extremely low. For example, patent CN202110197015.1 discloses a four-gene editing scheme related to pigs MSTN, SST, CD163 and pAPN, the core idea is to improve editing efficiency by modifying Cas9 high-efficiency expression vectors, but the scheme does not solve the problem of vector synergy of multi-gene synchronous editing, and still faces the challenges of unbalanced multi-gene editing efficiency and complex screening, meanwhile, part of multi-gene editing schemes are easy to cause problems of embryo dysplasia, individual physiological defects (such as MSTN single gene editing pig hindlimb weakness deformity) and the like due to improper target site selection or unreasonable editing strategy, so that the industrialized application of the technology is limited. The multi-character polymerization period is long and the universality is poor: related patents are combined with traditional hybridization breeding by fractional editing to polymerize polygenic traits, and the method disclosed in patent CN202310881137.1 for simultaneously knocking out CD163, pAPN and MSTN genes and improving the disease resistance and quality of pigs is technically characterized in that introduced commercial pig seeds and local pig seeds edited by the MSTN genes are obtained respectively, the MSTN gene-edited binary hybrid pigs are obtained by hybridization, the binary hybrid pigs are hybridized with the CD163 and pAPN binary hybrid pigs, and finally the three genes of the editing traits are gathered in offspring individuals by the traditional hybridization method. The method needs to be subjected to hybridization breeding for at least more than two generations, the cultivation period is up to several years, the hybridization combination of specific pig varieties is depended, the restriction of the variety genetic background is large, the universality is insufficient, and the requirements of diff