CN118360331A - Construction and application of PRKDC gene knockout immunodeficient animal model - Google Patents

Abstract

The invention belongs to the field of animal model construction, and particularly relates to a construction method and application of an animal model for PRKDC gene knockout, wherein the research before the problem finds that the traditional target first and second advanced exons can be adopted to obtain the PRKDC gene knockout animal, but can not obtain an immunodeficiency phenotype. The invention designs two sgRNAs pieces of large fragment knockout aiming at three exons #78 and #80 of a PRKDC gene kinase functional region to obtain PRKDC gene knockout rabbits with an immunodeficiency phenotype. The method comprises the steps of connecting a sgRNA sequence with a U6 promoter, preparing Cas9mRNA and gRNA for microinjection through steps of in vitro transcription and the like, mixing the two RNAs, injecting into a fertilized egg of a prokaryotic rabbit, obtaining a young rabbit through embryo transplantation, and identifying a rabbit model with PRKDC gene knocked out through PCR amplification and Sanger sequencing.

Description

Construction and application of PRKDC gene knockout immunodeficient animal model

Technical Field

The present invention belongs to the field of biomedicine, relates to the field of animal model construction, and specifically relates to an animal model in which the PRDKC gene is knocked out.

Construction method and application of physical model.

Background technique

PRKDC, as a gene encoding DNA-dependent protein kinase DNA-PKcs, plays an important role in the development of the immune system and DNA repair in mammalian cells. It is mainly involved in lymphocyte development, antibody V(D)J recombination, immunoglobulin and T cell receptor gene assembly, the establishment of central immune tolerance, and also plays an important role in maintaining telomere length and stability, which is essential for the maintenance of chromosome stability. Therefore, inactivation of the PRKDC gene in animals will lead to severe combined immunodeficiency (SCID).

The most commonly used SCID model is the immunodeficient mouse, but due to the large differences in the immune system and species between mice and humans, and the small size and difficulty in operation, there are certain limitations in the research. Therefore, the present invention selects rabbits with moderate size, longer lifespan, and immune systems more similar to humans as research objects, and destroys the rabbit PRKDC gene by CRISPR-Cas9 technology to construct an immunodeficient rabbit model. In the previous research of the inventors, it was found that the use of traditional targeting of the first and second front exons for the PRKDC gene does not make the PRKDC gene deletion obtain an immunodeficiency phenotype. The present invention designs two sgRNAs for the kinase functional region of its DNA-dependent protein kinase catalytic subunit (3619-3914aa), and uses double sgRNA to knock out its three exons No. 78, 79 and 80, a 590bp fragment.

Summary of the invention

The problem that the PRKDC gene (Gene ID: 100125348) cannot obtain an immunodeficiency phenotype by using the traditional targeting of the first and second front exons. The present invention designs two sgRNAs for the kinase functional region of the DNA-dependent protein kinase catalytic subunit (3619-3914aa), and uses double sgRNAs to knock out the 78th, 79th and 80th exons, a 590bp fragment. The invention provides an efficient and simple method for obtaining a PRKDC gene knockout rabbit model, in order to achieve a better immunodeficiency phenotype, and provide an ideal animal model for the pathogenesis of immunodeficiency diseases and the humanization of the immune system.

Based on the above purpose, the technical solution adopted by the present invention is as follows:

In some embodiments, the present invention provides a method for constructing a PRKDC gene knockout immunodeficient rabbit, characterized in that: the method comprises the step of knocking out the PRKDC gene of the rabbit.

In some embodiments, the knockout uses CRISPR/Cas9 technology to design dual sgRNA target sites for the PRKDC gene of rabbits. 1. In some embodiments, the dual sgRNA target sites are respectively directed to introns and/or exons of the PRKDC gene of rabbits.

2. In some embodiments, the method is characterized in that the introns are intron 77 and intron 80 of the PRKDC gene, respectively.

3. In some embodiments, the method is characterized in that the sgRNA sequence targeting intron 70 of the PRKDC gene is SEQ ID NO.1, and the sgRNA sequence targeting intron 80 of the PRKDC gene is SEQ ID NO.2.

4. In some embodiments, the method is characterized in that the exons are respectively exons 78 and 80 of the PRKDC gene.

5. In some embodiments, the method is characterized in that the sgRNA sequence for exon 78 of the PRKDC gene is selected from one of SEQ ID NO.3-5, and the sgRNA sequence for exon 80 of the PRKDC gene is selected from one of SEQ ID NO.6-8.

8. Another aspect of the present invention provides the use of the immunodeficient rabbits constructed by the method in the study of humanization of the immune system.

9. Another aspect of the present invention further provides a PRKDC gene knockout kit based on CRISPR-Cas9 gene knockout technology, characterized in that the PRKDC gene knockout kit comprises an sgRNA shown in any one of SEQ ID NO.1-8.

10. Another aspect of the present invention further provides the kit, characterized in that the kit also includes a PRKDC-gRNA in vitro transcription vector, the PRKDC-gRNA in vitro transcription vector uses the pU6-gRNA vector as a starting vector and contains a gRNA targeting the PRKDC gene; the gRNA is obtained by annealing the primers shown in SEQ ID NO.9 to SEQ ID NO.24.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention uses rabbits as animal models for constructing PRKDC gene knockout. Compared with rats and mice, rabbits have a moderate body size, a longer lifespan, and an immune system that is more similar to that of humans. Based on this, the present invention uses efficient CRISPR/Cas9 gene editing technology and microinjection technology, selects exons 78 and 80 as target sites, constructs a PRKDC knockout rabbit model, obtains a better immunodeficiency phenotype, and provides an ideal animal model for the pathogenesis of immunodeficiency diseases and the humanization of the immune system.

(2) In our previous study, we obtained a knockout rabbit with a 50 bp deletion and a frameshift mutation by targeting the first exon of the PRKDC gene with a single sgRNA. However, in our subsequent study, we found that the T and B lymphocytes of the homozygous rabbits with a 50 bp knockout developed normally and the immune system was not affected (Figure 1). Since the PRKDC gene is relatively large, it is possible that the gene mutation in the front may cause a frameshift mutation in PRKDC, but it only increases the length of the encoded protein without affecting its function. The present invention designs sgRNAs for the three exons of exon 78 and exon 80 of the kinase functional region of the PRKDC gene, the base length of the target site of the sgRNA is about 20bp, the sgRNA sequence is connected to the T7 promoter, an in vitro transcription vector is obtained, and an in vitro transcription kit is used to prepare the Cas9mRNA and gRNA used for microinjection with the constructed in vitro transcription vector as a template, and then the Cas9mRNA and gRNA are mixed and injected into the fertilized egg of a single-cell rabbit, and the rabbit model of PRKDC gene knockout is obtained by PCR amplification and Sanger sequencing, and the T and B lymphocytes in the blood and lymphatic tissues are found to be missing through flow cytometry and HE staining, with a typical immunodeficiency phenotype, while the front exon of the single sgRNA targeting PRKDC gene has no phenotype. The method for constructing an animal model of PRKDC gene knockout of the present invention is simple, efficient and can truly simulate the human immunodeficiency phenotype, can be used for immunodeficiency diseases, CDX, PDX and other tumor-related research, or an animal model constructed by a humanized animal model of the immune system.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the development of T and B lymphocytes in rabbits with 50bp PRKDC gene knockout

B281 represents the breeding number; WT represents wild type; HO represents homozygous

Figure 2 shows the design of PRKDC gene sgRNA

sg1 represents sgRNA1, sg2 represents sgRNA2, targeting introns 77 and 80 of the PRKDC gene respectively; sg3-5 represents sgRNA3-5, targeting exon 78 of the PRKDC gene; sg3-5 represents sgRNA6-8, targeting exon 80 of the PRKDC gene

Figure 3 shows the sequencing results of double sgRNA targeting rabbits

R stands for surrogate mother rabbit, R004-R008 stands for the surrogate mother rabbit number

Figure 4 shows the genotype analysis of the T vector cloned and sequenced rabbits

Figure 5 is the flow cytometry analysis of SCID rabbit lymphocytes

Figure 6 is a statistical analysis of the flow cytometry results of SCID rabbit lymphocytes

Figure 7 shows the thymus and spleen of a SCID rabbit

Figure 8 is HE staining section of thymus and spleen of SCID rabbit

Detailed ways

To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

Example 1

The process of constructing the PRKDC gene knockout animal model in this embodiment is as follows: two sgRNAs are designed for the three exons of exon 78 and exon 80 of the PRKDC gene, and the base length of the target site of the sgRNA is about 20 bp. The sgRNA sequence is connected to the TU promoter to obtain an in vitro transcription vector, and an in vitro transcription kit is used to prepare Cas9mRNA and gRNA for microinjection using the constructed in vitro transcription vector as a template. Then, the Cas9mRNA and gRNA are mixed and injected into a single-cell stage rabbit fertilized egg, and a PRKDC gene knockout rabbit model is obtained by PCR amplification and Sanger sequencing. The specific method is as follows:

1.1 Design of sgRNA for target site of rabbit PRKDC gene knockout

The DNA-dependent protein kinase DNA-PKcs encoded by the PRKDC gene is a protein with important biological functions. It is mainly involved in lymphocyte development, antibody V(D)J recombination, immunoglobulin and T cell receptor gene assembly, and the establishment of central immune tolerance. It is a key gene for regulating the immune system. The NCBI database shows that the rabbit PRKDC gene has 82 exons, a total length of 171,323bp, and a predicted protein length of 4028aa. For the knockout of most genes, sgRNA can be designed for the first few exons, because there is a high probability of obtaining frameshift mutations during the DNA repair process, resulting in the loss of the entire gene function. In previous studies, a single sgRNA was used to knock out a 50bp fragment for the first exon (not reported), but no corresponding phenotype was produced. Therefore, to ensure the real knockout of PRKDC gene, we designed 8 sgRNAs for the kinase functional region of its DNA-dependent protein kinase catalytic subunit (3619-3914aa) as shown in Table 1 and Figure 2, and used double sgRNA to knock out its three exons 78, 79 and 80, a 590bp fragment. The design of sgRNA can refer to the following Cas9 target site prediction website: CRISPOR (tefor.net). After the target site is selected, it needs to be compared with the whole rabbit genome sequence on NCBI to confirm the uniqueness of the target sequence and avoid off-target.

Table 1

1.2 Determine the corresponding primers for each sgRNA

The targeting recognition sequences of sgRNA for exons 78 and 80 of the PRKDC gene are shown in Table 2:

The primers in Table 2 were dissolved in ddH2O to 10 μM solution, annealed, heated at 98°C for 10 min, and naturally cooled to room temperature. The primers were then annealed and connected to the gRNA cloning backbone vector Px330 plasmid after BbsI digestion, connected, transformed, cloned and sequenced to obtain the correct PRKDC-gRNA in vitro transcription vector.

1.4 Preparation of sgRNA and in vitro transcription of mRNA

After the plasmid sequencing was successfully constructed, the sgRNA expression vector with cutting effect was amplified using T7-F: GAAATTAATACGACTCACTATA and T7-R: AAAAAAAGCACCGACTCGGTGCCAC, and the obtained sgRNA amplification product was purified using the Tiangen General DNA Product Purification Kit. The mRNA was obtained by in vitro transcription using the HiScribe T7 High-Efficiency RNA Synthesis Kit E2040, and 2 μL was taken to detect the concentration. At the same time, 2 μL was taken to analyze the stability of the product by 2% agarose electrophoresis, diluted, aliquoted, and stored at -80°C. The GeneArtTM CRISPR nuclease mRNA purchased from InvitrogenTM was diluted to 150 ng/μL and aliquoted in RNase-free centrifuge tubes for storage at -80°C.

1.5 Rabbit fertilized egg injection and in vitro embryo targeting efficiency detection

Female rabbits over 5 months old were selected as donors for superovulation treatment, and 100 IU PMSG was injected subcutaneously. After 96 hours, they were mated with male rabbits and 100 IU human chorionic gonadotropin (HCG) was injected intravenously to induce ovulation. 18 hours after fertilization, the donor rabbit was euthanized. The oviduct was recovered, the fertilized eggs were flushed with operating fluid, the embryo fertilization was observed under a microscope, and the embryos in the pronuclear stage were microinjected. The rabbit embryos were placed in the operating fluid covered with paraffin oil using a 120-150 μm diameter mouth pipette. Cytoplasmic injection was performed with a mixture containing 100 ng/μL Cas9mRNA and 50 ng/μL sgRNA or 100 ng/μL plasmid. After injection, the embryos were washed three times in the culture medium and cultured in an incubator under the conditions of 38.5 ° C, 5% CO2, and saturated humidity. After injection, the embryos used for in vitro testing were placed in oocyte maturation medium, cultured for 72 hours until the blastocyst stage was recovered, washed several times with PBS, and each embryo was placed in 5 μL of NP40 lysis solution, 56°C, 1 hour, and 96°C for 10 minutes. Genomic DNA was extracted for PCR amplification and sequencing. After sequencing, the combination of sgRNA4 (SEQ ID NO.4) and sgRNA6 (SEQID NO.6) was analyzed to obtain efficient gene knockout efficiency. Subsequent in vivo studies were performed using this pair of sgRNA combinations (as shown in Table 3).

table 3

1.6 Construction of PRKDC gene-edited rabbits

To obtain gene-edited rabbits, the injected embryos were placed in an incubator for a period of time and then transplanted into the oviduct of the recipient rabbit. 12-25 fertilized eggs were transplanted on each side. The transplantation, birth and gene knockout conditions are shown in Table 4. The surrogate rabbits are numbered R004-R008 and marked in red. The genome of all rabbit ear tissues was extracted using the Takara MiniBEST Universal Genomic DNA Extraction Kit, and the PRKDC gene was amplified by PCR. After electrophoresis detection, it was purified and sent for sequencing. The sequencing results were compared with the GeneBank reference sequence.

The results showed that among the 19 rabbits produced by plasmid injection, 3 offspring carried PRKDC gene mutation individuals. Among the offspring injected with mRNA, 7 offspring were homozygous knockout individuals, and the remaining 19 were heterozygous knockout individuals (as shown in Table 4).

In order to clarify the specific gene editing situation of each individual, we performed T-vector sequencing on the heterozygous knockout individuals with overlapping peaks, picked 5 single clones for sequencing each time, and sorted and analyzed the genotypes of the mRNA-injected rabbits (as shown in Figures 3 and 4).

Table 4

1.7 Phenotypic analysis of lymphocytes and tissue development

To analyze the changes in immune cell components in the whole blood of PRKDC knockout rabbits, about 1 mL of peripheral blood was collected from the central ear artery of the rabbit and placed in an anticoagulation blood collection tube for flow cytometry detection. For rabbits with PRKDC knockout identified, the development of the corresponding immune organs was identified and analyzed. The thymus and spleen of wild-type and double knockout rabbits were taken, placed in a 4% formaldehyde solution, and fixed overnight. The dehydrated samples were paraffin-embedded and sectioned, and HE staining was performed to observe the differences in tissue morphology.

We used flow cytometry to analyze the development of peripheral blood lymphocytes (as shown in Figures 5 and 6). R007-3 was a homozygous knockout rabbit, and R007-6 and R008-3 were heterozygous knockout rabbits carrying multiple mutations. It was found that the number of lymphocytes in homozygous and partially heterozygous SCID rabbits was significantly reduced. Among the 1×10 ⁴ cells counted, in WT rabbits (n=3), T cells (CD5 ⁺ ) accounted for 13.5%, B cells (IgM) accounted for 16.25%, CD4 ⁺ T cells accounted for 8.8%, CD8 ⁺ T cells accounted for 9.1%, and CD4 ⁺ /CD8 ⁺ T cells accounted for 0.7%. In SCID rabbits (n=4), T cells (CD5 ⁺ ) accounted for 0.025%, B cells (IgM) accounted for 0.175%, CD4 ⁺ T cells accounted for 0.025%, CD8+T cells accounted for 0.1%, and CD4 ⁺ /CD8 ⁺ T cells accounted for 0%. Statistical analysis ****P<0.0001 is considered to be extremely significant. From the statistical data, it can be concluded that the number of lymphocytes in SCID rabbits is significantly reduced compared with that in WT rabbits.

To observe the effect of PRKDC knockout on the development of immune organs, we dissected the rabbits and observed and counted the development of the thymus. It can be seen from Figure 7 that the thymus of PRKDC knockout rabbits was significantly atrophied or disappeared compared with WT rabbits; while the spleen did not change significantly in appearance. Subsequently, HE staining of relevant immune tissues was performed (Figure 8), and after pathological analysis of the sections, R008-4 was a homozygous knockout rabbit and R006-6 was a heterozygous knockout rabbit. The results showed that in the field of view of Figure (a), the thymus tissue structure was disordered, the volume was small, the boundary between the cortex and medulla was unclear, the number of lymphocytes was reduced, more vascular congestion was visible (black arrows), and a small amount of lymphocyte infiltration was visible in the lobular interstitium (red arrows). In the field of view of Figure (b), the spleen tissue showed a disordered structure, a small number of lymphocytes, and no obvious red pulp and white pulp structure. In Figures (b)-1 and 2, the connective tissue hyperplasia divides it into lobules, and many capillary congestion (black arrows) can be seen, small focal hemorrhages can be seen in the local interstitium (red arrows), and lymphocyte infiltration (yellow arrows) can be seen occasionally. Figure (b)-3.4 shows a lot of medullary sinus congestion (red arrows).

Claims (10)

1. A method for constructing a PRKDC gene knockout immunodeficient rabbit, characterized in that: the method comprises the step of knocking out the PRKDC gene of the rabbit. 2. The method according to claim 1, characterized in that: the knockout uses CRISPR/Cas9 technology to design dual sgRNA target sites for the PRKDC gene of rabbits. 3. The method according to claim 2, characterized in that: the dual sgRNA target sites are respectively directed to introns and/or exons of the PRKDC gene of rabbits. 4. The method according to claim 3, characterized in that the introns are intron 77 and intron 80 of the PRKDC gene respectively. 5. The method according to claim 4, characterized in that the sgRNA sequence for intron 70 of the PRKDC gene is SEQ ID NO.1, and the sgRNA sequence for intron 80 of the PRKDC gene is SEQ ID NO.2. 6. The method according to claim 3, characterized in that the exons are respectively exon 78 and exon 80 of the PRKDC gene. 7. The method according to claim 6, characterized in that the sgRNA sequence for exon 78 of the PRKDC gene is selected from one of SEQ ID NO.3-5, and the sgRNA sequence for exon 80 of the PRKDC gene is selected from one of SEQ ID NO.6-8. 8. Use of the immunodeficient rabbits constructed by the method according to any one of claims 1 to 7 in studies on humanization of the immune system. 9. A PRKDC gene knockout kit based on CRISPR-Cas9 gene knockout technology, characterized in that the PRKDC gene knockout kit comprises an sgRNA shown in any one of SEQ ID NO.1-8. 10. The kit according to claim 10, characterized in that the kit also includes a PRKDC-gRNA in vitro transcription vector, the PRKDC-gRNA in vitro transcription vector uses the pU6-gRNA vector as a starting vector and contains a gRNA targeting the PRKDC gene; the gRNA is obtained by annealing the primers shown in SEQ ID NO.9 to SEQ ID NO.24.