CN-122012401-A - Efficient enrichment of blue-ear-resistant high-fertility edited pig donor cells, and preparation method and application thereof

Abstract

The invention discloses a method for efficiently enriching blue-ear-resistant high-fertility editing pig donor cells, which comprises the steps of introducing a CRISPR-Cas9 plasmid targeting BMP15 genes, a CRISPR-Cas9 plasmid targeting CD163 genes and a double-gene editing alternative reporter plasmid into pig donor cells, culturing the pig donor cells to enable gene editing and reporter gene expression to occur, detecting and sorting cells positive in reporter gene expression, and thus obtaining the enriched double-gene editing donor cells, wherein the double-gene editing alternative reporter plasmid comprises a BMP15 gene editing target recognition sequence, a transcription terminator sequence, a CD163 gene editing target recognition sequence and a fluorescence reporter gene sequence which are sequentially connected. The invention constructs the substitution report plasmid for the BMP15 and CD163 double-gene editing, and can convert the editing condition of the target loci of the BMP15 and CD163 genes in the genome of the cells into a detectable red fluorescent phenotype, thereby improving the enrichment efficiency of the pig donor cells for resisting the blue-ear high-fertility editing and effectively improving the double-gene editing efficiency of the cells.

Inventors

• HE ZUYONG
• WANG RUI
• YANG CHANGQI
• HE JIAN
• CHEN YAOSHENG

Assignees

• 中山大学

Dates

Publication Date

20260512

Application Date

20260213

Claims (10)

1. 1. A method for efficiently enriching anti-blue-ear high-fertility edited pig donor cells, comprising the steps of: S1, introducing a CRISPR-Cas9 plasmid targeting BMP15 gene, a CRISPR-Cas9 plasmid targeting CD163 gene and a double-gene edited alternative reporter plasmid into a pig donor cell; s2, culturing the pig donor cells to enable gene editing and reporter gene expression to occur; s3, detecting and sorting cells positive for reporter gene expression, so as to obtain enriched double-gene editing donor cells; The alternative reporting plasmid for double gene editing comprises a recognition sequence of a BMP15 gene editing target, a transcription terminator sequence, a recognition sequence of a CD163 gene editing target and a DsRed reporting gene sequence which are connected in sequence.
2. 2. The method of claim 1, wherein the BMP15 gene editing target is identified by SEQ ID No. 1.
3. 3. The method of claim 1, wherein the transcription terminator sequence is set forth in SEQ ID NO. 2.
4. 4. The method of claim 1, wherein the recognition sequence of the CD163 gene editing target is shown in SEQ ID NO. 3.
5. 5. The method of claim 1, wherein the DsRed reporter gene sequence is set forth in SEQ ID No. 4.
6. 6. The method of claim 1, wherein the CRISPR-Cas9 plasmid targeting BMP15 gene contains sgrnas targeting BMP15 gene, the nucleotide sequence of which is shown in SEQ ID No. 6.
7. 7. The method of claim 1, wherein the CD163 gene-targeting CRISPR-Cas9 plasmid contains sgrnas targeting BMP15 gene, the nucleotide sequence of which is shown in SEQ ID No. 7.
8. 8. A dual-gene editing alternative report plasmid is characterized by comprising the following elements of a BMP15 gene editing target recognition sequence, a transcription terminator sequence, a CD163 gene editing target recognition sequence and a DsRed report gene sequence which are connected in sequence.
9. 9. A kit for enriching for double gene editing cells comprising the double gene editing alternative reporter plasmid of claim 8.
10. 10. Use of the dual gene edited alternative reporter plasmid of claim 8, the kit of claim 9 for enriching anti-blue-ear high fertility editing pig donor cells.

Description

Efficient enrichment of blue-ear-resistant high-fertility edited pig donor cells, and preparation method and application thereof Technical Field The invention belongs to the technical field of genetic engineering biology, and particularly relates to an efficient enrichment blue-ear-resistant high-fertility editing pig donor cell, and a preparation method and application thereof. Background In preparing a dual gene editing pig model for xenogeneic organ transplantation or biomedical research, obtaining high quality, precisely edited donor cells is a critical first step. Currently, the mainstream technical route in the field is to use porcine primary cells (such as fetal fibroblasts) as starting materials and realize the synchronous editing of double gene loci by transfecting a plasmid vector containing a CRISPR-Cas9 system and two specific guide RNAs (gRNAs). In specific implementation, the primary cells of the pig are usually isolated and cultured first, and after the cell state is stable, two plasmids respectively carrying the Cas9 protein expression frame and the specific gRNA sequence are introduced into the cells by using liposome package transfection or electroporation transfection and other technologies. After transfection, the cells were cultured in an incubator for a period of time sufficient for CRISPR-Cas9 system expression and complete gene cleavage and editing. These treated cells were then collected as a donor nucleus source for subsequent Somatic Cell Nuclear Transfer (SCNT) in the hope of ultimately obtaining a well-defined double gene edited pig. However, this seemingly clear experimental path still faces a series of technical bottlenecks in the actual operation, resulting in often unsatisfactory efficiency in the final acquisition of the desired donor cells. The most prominent problem is that the proportion of cells obtained for simultaneous editing of the double genes is generally low. This is mainly caused by the fact that, firstly, the cell transfection efficiency itself is difficult to reach 100%, and both the liposome method and the electrotransfer method have a certain proportion of cells which cannot successfully introduce exogenous plasmids, and the cells cannot naturally undergo any editing. Second, even if the plasmid successfully enters the cell, there is often a difference in cleavage activity between the two grnas, which may result in efficient editing of one gene locus and low editing efficiency of the other locus, thereby reducing the probability of occurrence of a double editing event. In addition, the DNA repair mechanisms of the cells themselves (e.g., non-homologous end joining or homologous recombination repair) are randomized and may produce unintended editing results. Meanwhile, the primary cells have limited proliferation capacity and are sensitive to external transfection stimulus, and the survival state of the primary cells also directly influences editing effect. These factors are superimposed on each other, so that cell cloning, in which screening of the double genes in the cell population all achieves the desired modification (such as double allele knockout), becomes extremely laborious and costly, and becomes one of the major obstacles restricting the large-scale application of the technology. Therefore, developing more efficient co-transfection strategies, optimizing gRNA design and enhancing cell screening methods remains a breakthrough in the art. Disclosure of Invention The invention aims at solving the technical problems and provides a method for efficiently enriching anti-blue-ear high-fertility editing pig donor cells. In order to achieve the above purpose, the present invention provides the following technical solutions: In a first aspect, the invention provides a method for efficient enrichment of anti-blue-ear high fertility editing pig donor cells, comprising: S1, introducing a CRISPR-Cas9 plasmid targeting BMP15 gene, a CRISPR-Cas9 plasmid targeting CD163 gene and a double-gene edited alternative reporter plasmid into a pig donor cell; s2, culturing the pig donor cells to enable gene editing and reporter gene expression to occur; s3, detecting and sorting cells positive for reporter gene expression, so as to obtain enriched double-gene editing donor cells; the alternative reporting plasmid for double gene editing comprises a recognition sequence of a BMP15 gene editing target, a transcription terminator sequence, a recognition sequence of a CD163 gene editing target and a fluorescent reporting gene sequence which are connected in sequence. Preferably, the recognition sequence of the BMP15 gene editing target is shown as SEQ ID NO. 1. Preferably, the transcription terminator sequence is shown in SEQ ID NO. 2. Preferably, the identification sequence of the CD163 gene editing target is shown as SEQ ID NO. 3. Preferably, the fluorescent reporter is a DsRed reporter. Preferably, the DsRed reporter gene sequence is shown in SEQ ID NO. 4. Preferably, the se