Construction method of congenital pure red cell anemia zebra fish model

Abstract

The application relates to a method for constructing a congenital pure red cell anemia zebra fish model, which comprises the following steps: performing directional mutation on a bysl gene in a wild-type zebra fish embryo to obtain F0 generation zebra fish; mating the F0 generation zebra fish with wild-type zebra fish to screen positive heterozygote F1 generation zebra fish from born zebra fish; mating the positive heterozygote F1 generation zebra fish with wild-type zebra fish to screen positive heterozygote F2 generation zebra fish from born zebra fish; and mating the positive heterozygote F2 generation zebra fish to screen positive homozygote zebra fish from born zebra fish as a congenital pure red cell anemia zebra fish model. The model can simulate the symptoms of congenital pure red cell anemia, and can be used for mechanism research, drug evaluation and new drug screening of the disease.

Description

Technical Field

[0001] This application relates to the field of genetic engineering, specifically to a method for constructing a zebrafish model of congenital pure red blood cell aplastic anemia. Background Technology

[0002] Diamond-Blackfan anemia (DBA), also known as congenital pure red blood cell aplasia, is one of the rare diseases listed in the first batch of rare diseases published by the National Health Commission of China in 2018. Its main characteristics include congenital erythrocyte hypoplasia (anemia), congenital malformations, and a high incidence of hematologic malignancies. DBA is caused by mutations in ribosome-related genes. The ribosome gene RPS19 was first reported to be associated with DBA in 1999, and it is also the gene with the highest mutation frequency in DBA patients, accounting for approximately 25%. Other mutations include RPS24, RPS17, RPL35A, and 22 other genes. However, currently, approximately 20%-25% of patients cannot be diagnosed with the mutated gene. The diverse pathogenic genes correspond to diverse pathogenic mechanisms. However, current animal models of congenital pure red cell aplasia only involve a limited number of genes, such as RPS19, RPL11, RPL5, RPS24, RPS7, RPS20, and GATA1, which is insufficient to explain the complexity of the pathogenesis of congenital pure red cell aplasia. Furthermore, they cannot meet the needs for targeted drug screening for different types of congenital pure red cell aplasia.

[0003] Therefore, it is necessary to construct a new animal model of congenital pure red blood cell aplastic anemia. Summary of the Invention

[0004] Based on this, one embodiment of this application provides a method for constructing a zebrafish model of congenital pure red blood cell aplastic anemia.

[0005] The technical solution includes:

[0006] A method for constructing a zebrafish model of congenital pure red blood cell aplasia includes:

[0007] Directed mutation of the bysl gene in wild-type zebrafish embryos yielded F0 generation zebrafish.

[0008] The F0 generation zebrafish were mated with wild-type zebrafish, and positive heterozygous F1 generation zebrafish were selected from the offspring.

[0009] The positive heterozygous F1 generation zebrafish were mated with wild-type zebrafish, and positive heterozygous F2 generation zebrafish were selected from the offspring; and,

[0010] The male and female positive heterozygous F2 generation zebrafish were hybridized, and positive homozygous zebrafish were selected from the offspring to serve as a zebrafish model of congenital pure red blood cell aplastic anemia.

[0011] In one embodiment, directing the mutation of the bysl gene in wild-type zebrafish embryos includes the following steps:

[0012] Prepare sgRNA targeting exon 1 of the zebrafish bysl gene, the sequence of which is shown in SEQ ID NO.1 and SEQ ID NO.2;

[0013] To prepare a Cas9 / sgRNA mixture, the sgRNA and Cas9 protein are mixed; and

[0014] The Cas9 / sgRNA mixture was introduced into wild-type zebrafish embryos.

[0015] In one embodiment, the Cas9 / sgRNA mixture was introduced into wild-type zebrafish embryos via microinjection.

[0016] The construction methods described above, including screening for positive heterozygous F1 generation zebrafish, screening for positive heterozygous F2 generation zebrafish, and screening for positive homozygous zebrafish, are independently selected from PCR and Sanger sequencing.

[0017] In one embodiment, the PCR method uses the detection primer pairs shown in SEQ ID NO.6~SEQ ID NO.7.

[0018] The method for constructing a zebrafish model of congenital pure red blood cell aplastic anemia describes the tissues or organs of the zebrafish model of aplastic anemia.

[0019] A kit for constructing a zebrafish model of congenital pure red blood cell aplastic anemia includes an sgRNA targeting exon 1 of the zebrafish bysl gene; the sequence of the sgRNA is shown in SEQ ID NO.1~SEQ ID NO.2.

[0020] In one embodiment, it also includes one or more of the following: Cas9 protein, PCR reaction buffer, and PCR reaction solution.

[0021] In one embodiment, the PCR reaction solution includes Taq DNA polymerase, dNTPs, and Mg. 2+ One or more of them.

[0022] The application of the zebrafish model of congenital pure red blood cell aplastic anemia constructed by the described method in drug screening.

[0023] Compared with traditional technologies, this application has the following advantages:

[0024] This application constructs a zebrafish model of congenital pure red cell aplastic anemia based on bysl gene knockout, which simulates the symptoms of congenital pure red cell aplastic anemia and shares the same pathogenic basis as the disease. This model can not only be used for screening and confirming new pathogenic genes of congenital pure red cell aplastic anemia, but also for mechanism research, drug evaluation and new drug screening of the disease. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The predicted results for the gene structure (Figure A), sgRNA target location (Figure A), mutation form (Figure B), and protein translation (Figure C) of bysl.

[0027] Figure 2 The homozygous mutant of bysl zebrafish exhibits head and eye dysplasia (Fig. B) and cardiac edema (Fig. A).

[0028] Figure 3 The results of in situ hybridization experiments using CMYB (Figure A) and hbae1.1 (Figure B) RNA probes to label hematopoietic stem cells and erythrocytes, respectively.

[0029] Figure 4 The expression of p53 and its downstream molecules mdm2, p21, ccng1, casp8, and casp10 in the homozygous bysl mutant was detected by qPCR.

[0030] Figure 5 Abnormal ribosome synthesis was observed in the homozygous mutant of bysl zebrafish; Figure A shows the transcriptome analysis results, Figure B shows the qPCR analysis results, and Figures C and D show the capillary electrophoresis analysis results. Detailed Implementation

[0031] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided below. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0033] The term “and / or” as used herein includes any and all combinations of one or more of the related listed items.

[0034] The main English explanations used in this article are as follows:

[0035] CRISPR / Cas9: Clustered Regularly Interspersed Short Palindromic Repeats / Cas9 is a gene editing system that utilizes the Cas9 protein.

[0036] DPF: Days Post-Fertilization.

[0037] rRNA: ribosomal RNA, is the backbone structure of the ribosome.

[0038] sgRNA: single guide RNA, used in combination with Cas9 protein, can guide Cas9 protein to its target DNA sequence for editing.

[0039] qPCR: Quantitative Real-time PCR, is a method for real-time monitoring and analysis of PCR amplification products.

[0040] cDNA: complementary DNA, specifically refers to a DNA strand that is reverse transcribed in vitro and becomes complementary to RNA.

[0041] The Bysl gene is highly conserved in eukaryotes and has been shown to regulate the processing of 18S ribosomal RNA, thereby participating in the in vivo synthesis of ribosomes. In tumor cells and mice, Bysl can regulate cell growth and embryonic development. Currently, there are no reported animal models using the CRISPR / Cas9 system to knock out the Bysl gene to study its function in hematopoietic system development. Zebrafish are an excellent model organism for studying hematopoietic system development. Their hematopoietic development process is similar to that of humans, and zebrafish embryos develop in vitro, are transparent, and have transgenic strains with various blood cell lineages, facilitating direct observation of blood cell growth, differentiation, and migration using imaging techniques. In addition, zebrafish have high spawn production and low breeding costs, making them suitable for drug screening. Therefore, zebrafish models are becoming increasingly popular in the study of the mechanisms of hematopoietic diseases and in drug screening.

[0042] One embodiment of this application provides a method for constructing a zebrafish model of congenital pure red blood cell aplasia, including:

[0043] Directed mutation of the bysl gene in wild-type zebrafish embryos yielded F0 generation zebrafish.

[0044] The F0 generation zebrafish were mated with wild-type zebrafish, and positive heterozygous F1 generation zebrafish were selected from the offspring.

[0045] The positive heterozygous F1 generation zebrafish were mated with wild-type zebrafish, and positive heterozygous F2 generation zebrafish were selected from the offspring; and

[0046] The male and female positive heterozygous F2 generation zebrafish were hybridized, and positive homozygous zebrafish were selected from the offspring to serve as a zebrafish model of congenital pure red blood cell aplastic anemia.

[0047] In one specific example, the bysl gene was directed to be mutated in wild zebrafish embryos using the CRISPR / Cas9 system to obtain zebrafish mutants of bysl, which simulated the symptoms of congenital pure red blood cell aplastic anemia. This method has the advantages of short cycle, stability, low cost and good reproducibility.

[0048] Directed mutation of the bysl gene in wild-type zebrafish embryos can reduce the expression of the bysl gene or protein and / or inactivate its function.

[0049] Specifically, the mutation of the bysl gene in wild-type zebrafish embryos includes the following steps: preparing sgRNA targeting exon 1 of the zebrafish bysl gene, the sequence of which is shown in SEQ ID NO.1 and SEQ ID NO.2; mixing the sgRNA with Cas9 protein to prepare a Cas9 / sgRNA mixture; and introducing the Cas9 / sgRNA mixture into wild-type zebrafish embryos.

[0050] In one specific example, the Cas9 / sgRNA mixture was introduced into wild-type zebrafish embryos via microinjection.

[0051] In one specific example, PCR and Sanger sequencing were used to screen for positive heterozygous F1 generation zebrafish, or positive heterozygous F2 generation zebrafish, or positive homozygous zebrafish.

[0052] In one specific example, the PCR method uses the detection primer pairs shown in SEQ ID NO.6~SEQ ID NO.7.

[0053] One embodiment of this application also provides tissues or organs of the zebrafish model of aplastic anemia established by the above-described method for constructing a zebrafish model of congenital pure red blood cell aplastic anemia.

[0054] One embodiment of this application also provides a kit for constructing a zebrafish model of congenital pure red blood cell aplastic anemia, including sgRNA targeting exon 1 of the zebrafish bysl gene.

[0055] In one specific example, the sequence of the sgRNA is shown in SEQ ID NO.1 and SEQ ID NO.2.

[0056] In a specific example, it also includes one or more of the following: Cas9 protein, PCR reaction buffer, and PCR reaction solution.

[0057] In one specific example, the PCR reaction solution includes Taq DNA polymerase, dNTPs, and Mg. 2+ One or more of them.

[0058] The construction method described in this application not only provides a new animal model for the study of the mechanism of congenital pure red cell aplastic anemia, which helps to reveal the screening and evidence of new pathogenic genes, but also provides a stable, in vivo, reproducible, high-success-rate, and low-cost animal model for drug screening of congenital pure red cell aplastic anemia.

[0059] An embodiment of this application also provides the application of the zebrafish model of congenital pure red blood cell aplastic anemia constructed by the above-described construction method in drug screening.

[0060] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0061] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0062] Example 1

[0063] I. Model Construction

[0064] 1. Determine the sequence of the sgRNA targeting bysl.

[0065] To knock out the zebrafish bysl gene, the CRISPRscan website (https: / / www.crisprscan.org / ) was used to predict sgRNAs targeting bysl. To improve the efficiency of bysl knockout, two sgRNAs were selected and named bysl sgRNA #1 and bysl sgRNA #2, respectively. The target sequences of these two sgRNAs are sgRNA #1: 5'-GTGTCCTGCGCTCCTCCT-3' (SEQ ID NO.1); sgRNA #2: 5'-GATGAGAAGCTGAGCCGG-3' (SEQ ID NO.2). Both sgRNA target sequences are located on exon 1 of the bysl gene and are located in the coding region of bysl. Subsequently, primers for in vitro synthesis of sgRNAs were synthesized according to the target sequences of the sgRNAs (all primers were ordered from Beijing Qingke Biotechnology Co., Ltd.), and the primer sequences are as follows:

[0066] bysl sgRNA #1 fw (SEQ ID NO.3):

[0067] 5'-taatacgactcactataGGGTGTCCTGCGCTCCTCCTgttttagagctagaa-3';

[0068] bysl sgRNA #2 fw (SEQ ID NO.4):

[0069] 5'-taatacgactcactataGGGATGAGAAGCTGAGCCGGgttttagagctagaa-3';

[0070] Universal primer Tail primer (SEQ ID NO.5):

[0071] 5'-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'.

[0072] 2. Synthesize the DNA template for bysl sgRNA.

[0073] The obtained sgRNA primers were added to an appropriate amount of distilled water according to the instructions to make the primer concentration 10 mM. Then, the PCR system was prepared according to the proportions shown in Table 1.

[0074] Table 1

[0075]

[0076] The reaction mixture was then placed in a PCR instrument (Thermo Fisher, catalog number: 4375786) and reacted according to the procedure shown in Table 2:

[0077] Table 2

[0078]

[0079] After the reaction, the product was detected by agarose gel electrophoresis, and it should be a single band of approximately 117 bp (base pair). The product was then purified using a PCR purification kit (Yisheng Biotechnology (Shanghai) Co., Ltd., catalog number: 19106ES50). The product concentration was then measured using a NanoDrop micro-spectrophotometer (Thermo Fisher, catalog number: ND-2000).

[0080] 3. Synthesis of sgRNA

[0081] Transcription was performed using the T7 RNA transcription kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number: TR101-01) with purified PCR products as templates, following the manufacturer's instructions. After transcription, the transcription products were detected by agarose gel electrophoresis; the product should be a single band. Subsequently, the sgRNA was purified by adding 1 / 10 volume of ammonium acetate solution (5 M) to the transcription system, followed by adding water to a total volume of 150 μl. Then add an equal volume of phenol-chloroform-isoamyl alcohol mixture (Sigma Aldrich, catalog number: 77617), vortex thoroughly, and centrifuge at 12000 g for 30 seconds to separate the liquid into layers. Aspirate the upper aqueous phase and transfer it to a new centrifuge tube. Add 1150 μl of chloroform, vortex thoroughly, and centrifuge again at 12000 g for 30 seconds to separate the liquid into layers. Aspirate the upper aqueous phase and transfer it to a new centrifuge tube. Add 150 μl of isopropanol, mix well, and place in a -80°C freezer for at least 30 minutes. Then, centrifuge at 12000 g for 15 minutes in a centrifuge pre-cooled to 4°C. Discard the supernatant and retain the precipitate. Add 1 ml of 70% ethanol, centrifuge again at 12000 g for 15 minutes, discard the supernatant, and retain the precipitate. Then, open the cap and allow to stand at room temperature for the alcohol to evaporate (approximately 5 minutes). Add 20 μl of DNase- and RNase-free pure water. The concentration of sgRNA was detected using a NanoDrop micro-spectrophotometer and stored at -80°C.

[0082] 4. Construction of F0 generation bysl mutant zebrafish

[0083] A mixture of Cas9 protein (GenScript Biotechnology Co., Ltd., catalog number: Z03469) and sgRNA was prepared to achieve a final concentration of 100 ng / μl for Cas9 protein and 50 ng / μl for sgRNA. Then, using a microinjector (Harvard Apparatus, catalog number: PLI-100A), 2 nl of the mixture was injected into each wild-type embryo (one-cell stage) at the yolk sac. After injection, the embryos were allowed to develop to 24 hpf (hours postfertilization), and 10 embryos were collected. After aspiration, 100 μl of NaOH (50 mM) was added, and the embryos were incubated in a 95°C metal bath for 20 minutes. Then, 10 μl of Tris pH 7.5 (10 mM) was added to neutralize the lysis buffer. The mixture was then used as the DNA template for PCR. The primers used to detect the bysl target mutation were bysl fw: 5'-CGCGATTTCACACTCATAATT-3' (SEQ ID NO. 6) and bysl rv: 5'-AGTCAGTGAGTGCAGCATTCA-3' (SEQ ID NO. 7). After PCR, the PCR products were detected by agarose gel electrophoresis, and the PCR product size should be 326 bp. Subsequently, the PCR products were subjected to Sanger sequencing (sequencing service provided by Beijing Qingke Biotechnology Co., Ltd.), using bysl fw primers. After the bysl gene is successfully knocked out, the sequencing results should show different, disordered sequences starting from the target site. Finally, the remaining embryos were raised to adulthood.

[0084] 5. Screening of F0 generation BySL mutant zebrafish

[0085] To screen for F0 individuals containing the heritable bysl mutant allele, sexually mature F0 individuals were mated with wild-type individuals, and the resulting embryos were collected. Ten embryos were then lysed as DNA templates for PCR, and PCR was performed using bysl fw and bysl rv primers. The PCR products were detected by agarose gel electrophoresis, followed by Sanger sequencing. Embryos showing multiple sequences at the target site were considered to have parentages of bysl mutant F0 individuals. The remaining embryos were then raised to adulthood, becoming the F1 generation of bysl mutant zebrafish.

[0086] 6. Screening of F1 generation BySL mutant zebrafish

[0087] To identify F1 individuals with the bysl mutation, we used pointed forceps to collect 2-3 scales from the lateral sides of different F1 individuals. These scales were placed in centrifuge tubes, and 20 μl of NaOH (50 mM) was added. The tubes were incubated in a 95°C metal bath for 20 minutes, followed by the addition of 10 μl of Tris pH 7.5 (10 mM) to neutralize the lysis buffer. The mixture was then used as the DNA template for PCR. PCR was performed using bysl fw and bysl rv primers. The PCR products were detected by agarose gel electrophoresis and then Sanger sequencing. By comparing the sequence with existing bysl gene sequences (from the National Center for Biotechnology Information database, also known as the NCBI database, https: / / www.ncbi.nlm.nih.gov / , Gene ID: 394081), the mutation form of bysl was identified. Mutations where the number of inserted or deleted base pairs was not a multiple of three resulted in a frameshift in the mRNA translation reading frame, and were therefore considered effective knockout forms. Among them, a mutation with a 58 bp deletion was identified. Subsequently, this heterozygous mutant was crossbred with wild-type zebrafish for propagation, and the F2 generation of bysl mutant zebrafish was screened and identified using the same method.

[0088] 7. Morphological and developmental phenotypic analysis of bysl homozygous mutants

[0089] To analyze the impact of complete bysl knockout on embryonic development, heterozygous bysl mutants were mated, and homozygous bysl mutants were obtained through genotyping. The development of organs such as the head, eyes, and heart in the embryos was then analyzed under a microscope. These organs are commonly affected in patients with congenital pure red cell aplasia. Analysis revealed that the homozygous bysl mutant exhibited morphological developmental defects including head and eye dysplasia, and pericardial edema.

[0090] 8. Analysis of hematopoietic stem cells and erythrocytes of homozygous bysl mutants

[0091] Congenital pure red blood cell aplasia is characterized by a reduction in hematopoietic stem cells and anemia; therefore, the hematopoietic stem cells and erythrocytes of the bysl homozygous mutant were analyzed. Whole-embryo in situ RNA hybridization (WICH) was employed, using CMYB (labeling hematopoietic stem cells) and hbae1.1 (labeling erythrocytes) RNA probes for labeling, followed by microscopic imaging. The experimental steps of WICH were as follows: First, CMYB and hbae1.1 RNA probes were synthesized according to the instructions using the T7 RNA transcription kit and DIG RNA labeling mixture (Roche, catalog number: 11277073910). These probes were then purified according to the instructions and diluted to a concentration of 1 ng / μl with prehybridization buffer (50% formamide, 500 μg / ml yeast RNA, 50 μg / ml heparin, 5× SSC buffer). Embryos at 4 days post-fertilization (dpf) were then collected, fixed overnight at 4°C with 4% paraformaldehyde, and rinsed three times with PBST (PBS buffer containing 0.1% Tween-20) for 5 minutes each time. Proteinase K solution (20 μg / ml, Thermo Fisher, catalog number: EO0491) was then added to the embryos, and the embryos were placed in a 37°C metal bath for 35 minutes. Fixation was then performed again with 4% paraformaldehyde at room temperature for 20 minutes, followed by three rinses with PBST for 5 minutes each time. Finally, prehybridization solution was added, and the embryos were incubated at 60°C for 2 hours. The prehybridization solution was then removed, the probe was added, and the embryos were incubated overnight at 60°C. On the second day, the embryos were incubated twice at 60°C for 30 minutes each time using wash buffer 1 (50% formamide, 4× SSC solution, 0.1% Tween-20), wash buffer 2 (4× SSC solution, 0.1% Tween-20), and wash buffer 3 (0.4× SSC solution, 0.1% Tween-20). Then, the embryos were rinsed three times at room temperature with maleic acid buffer (MAB, pH 7.5, containing 0.1% Tween-20), followed by incubation with blocking solution (4% BSA solution) for 2 hours at room temperature. Finally, the embryos were incubated overnight at 4°C with anti-DIG-AP solution (1:2500 dissolved in 4% BSA solution, Roche, catalog number: 11093274910). Finally, the embryos were rinsed three times with PBST for 30 minutes each time, and incubated for 10 minutes with equilibration buffer (25 mM NaOH, 25 mM Tris-HCl pH 9.5, 0.1% Tween-20, 50 mM MgCl2). Finally, the embryos were developed in NBT / BCIP chromogenic solution (1:50 diluted in equilibration buffer, Roche, catalog number: 11681451001) until the signal was strong enough. The reaction was terminated with PBST and the background color was removed with 70% ethanol.

[0092] In situ hybridization results showed that the number of CMYB-labeled hematopoietic stem cells and HBAE1.1-labeled erythrocytes was significantly reduced in the bysl homozygous mutant.

[0093] 9. Detection of the p53 signaling pathway in the bysl homozygous mutant

[0094] A characteristic feature of congenital pure red cell aplasia is the activation of the p53 signaling pathway. Therefore, qPCR was used to detect the expression level of the p53 signaling pathway in homozygous BySL mutants. First, 4-day-fiber (dpf) BySL homozygous mutant embryos and control embryos were collected, and total RNA was extracted using TRIzol reagent (Thermo Fisher, catalog number: 15596026) according to the manufacturer's instructions. Subsequently, a cDNA library was synthesized using a reverse transcription kit (Takara, catalog number: RR047Q). Finally, qPCR reaction was performed using qPCR premix (Beijing TransGen Biotech Co., Ltd., catalog number: AQ602) according to the manufacturer's instructions. The real-time quantitative PCR instrument used was a LightCycler® 480 (Roche, catalog number: 05015278001). Primers for detecting the p53 gene and downstream genes, including mdm2, p21, ccng1, casp8, and casp10, are shown in Table 3.

[0095] Table 3

[0096]

[0097] The results of qPCR showed that the expression of p53 and its downstream genes was significantly upregulated, and the p53 signaling pathway was activated.

[0098] 10. Transcriptome analysis of homozygous bysl mutants

[0099] To better verify the similarity between the homozygous BySL mutant and congenital pure red cell aplasia, 4-day-fiber (dpf) BySL homozygous mutant and control embryos were collected and transcriptome sequencing was performed. Sample preparation, sequencing experiments, and data analysis were all performed by Beijing Novogene Technology Co., Ltd. KEGG signaling pathway analysis revealed that the ribosome signaling pathway was significantly upregulated in the BySL homozygous mutant. Abnormal ribosome synthesis is a pathogenic factor in congenital pure red cell aplasia. To further verify this result, qPCR was used to detect ribosome gene expression. The primers used for qPCR are shown in Figure 4.

[0100] Table 4

[0101]

[0102] Transcriptome sequencing and qPCR results showed that the expression of ribosome processing-related genes was significantly upregulated in the bysl homozygous mutant.

[0103] 11. Detection of ribosomal RNA from homozygous bysl mutants

[0104] Ribosomal RNA forms the structural backbone of ribosomes, and its content and ratio can reflect the normality of ribosome biosynthesis. Therefore, at 4 days post-fertilization (dpf) of embryonic development, total RNA was extracted from the homozygous bysl mutant and the control group. The total RNA samples were then subjected to capillary electrophoresis and quantification using an Agilent 2100 bioanalyzer to obtain the content of 28S and 18S molecules and calculate their ratio. In the control group, the 28S / 18S ratio was approximately 1.7, while in the homozygous bysl mutant, this ratio was approximately 3.2, indicating that ribosome processing in the homozygous bysl mutant is abnormal.

[0105] II. Result Verification:

[0106] 1. Gene structure, sgRNA target location, mutation types, and protein translation prediction of bysl

[0107] like Figure 1 As shown, zebrafish bysl has 7 exons, and the sgRNA target sites #1 and #2 designed in this application are located on exon 1. Figure 1 (A). By knocking out bysl using the CRISPR / Cas9 system, the bysl mutation obtained in this application involves the deletion of 58 base pairs ( Figure 1 (B). The mutant form of bysl is expected to encode a truncated 79-amino acid polypeptide chain, with the mutation occurring starting at amino acid 52, while the wild-type bysl gene encodes 422 amino acids. Figure 1 (C)

[0108] 2. The homozygous mutant of the bysl zebrafish exhibits underdeveloped head and eyes, as well as cardiac edema.

[0109] like Figure 2 As shown, morphological analysis of BySL zebrafish homozygous mutant embryos at 2 dpf, 4 dpf, and 5 dpf revealed that, compared to control embryos, their heads and eyes showed poor development starting at 2 dpf, and significant pericardial edema was also observed at 5 dpf. Figure 2 (A). Furthermore, its craniofacial cartilage is significantly absent ( Figure 2 (B)

[0110] 3. The bysl zebrafish homozygous mutant exhibits reduced hematopoietic stem cell count and anemia.

[0111] like Figure 3As shown, in situ hybridization experiments were performed using CMYB and HBAE1.1 RNA probes to label hematopoietic stem cells and erythrocytes, respectively. It was found that at 4 dpf, the number of hematopoietic stem cells and erythrocytes in the BySL homozygous mutant was significantly reduced. This is consistent with the characteristics of congenital pure red cell aplasia. Figure 3 (A and B in the middle).

[0112] 4. Activation of the p53 signaling pathway in the bysl zebrafish homozygous mutant

[0113] like Figure 4 As shown, qPCR was used to detect the expression of p53 and its downstream genes mdm2, p21, ccng1, casp8, and casp10 in the homozygous bysl mutant. The results showed that the expression of these genes was significantly upregulated in the bysl homozygous mutant. This indicates that the expression of p53 and its downstream genes was significantly upregulated, and the p53 signaling pathway was activated. Activation of the p53 signaling pathway is also one of the characteristics of congenital pure red cell aplasia.

[0114] 5. Abnormal ribosome synthesis was observed in the bysl zebrafish homozygous mutant.

[0115] like Figure 5 As shown, transcriptome analysis revealed a significant upregulation of the ribosome synthesis signaling pathway in the bysl zebrafish homozygous mutant. Figure 5 (A). Further qPCR experiments confirmed that genes related to ribosome synthesis, such as wdr43, rpp30, nmd3, utp4, snu13b, eif6, imp4, rrp7a, and csnk2a4, were significantly upregulated. Figure 5 (B). Subsequent capillary electrophoresis analysis revealed an abnormal 28S / 18S ratio in the bysl homozygous mutant. Figure 5 The 28S and 18S ribosomes are the backbone structures of the ribosome, indicating that the ribosome synthesis of the bysl homozygous mutant is disordered. Abnormal ribosome synthesis is the cause of congenital pure red blood cell aplastic anemia.

[0116] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0117] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing a zebrafish model of congenital pure red blood cell aplastic anemia based on bysl gene knockout, characterized in that, include: Directed mutation of the bysl gene in wild-type zebrafish embryos yielded F0 generation zebrafish. The F0 generation zebrafish were mated with wild-type zebrafish, and positive heterozygous F1 generation zebrafish were selected from the offspring. The positive heterozygous F1 generation zebrafish were mated with wild-type zebrafish, and positive heterozygous F2 generation zebrafish were selected from the offspring. as well as, The male and female zebrafish of the positive heterozygous F2 generation were hybridized, and positive homozygous zebrafish were selected from the offspring to serve as a zebrafish model of congenital pure red blood cell aplastic anemia. Directed mutation of the bysl gene in wild-type zebrafish embryos involves the following steps: Prepare sgRNA targeting exon 1 of the zebrafish bysl gene, wherein the targeting sequence of the sgRNA is shown in SEQ ID NO.1 and SEQ ID NO.2; To prepare a Cas9 / sgRNA mixture, the sgRNA and Cas9 protein are mixed; and The Cas9 / sgRNA mixture was introduced into wild-type zebrafish embryos via microinjection.

2. The construction method according to claim 1, characterized in that, The methods used to screen for positive heterozygous F1 generation zebrafish, positive heterozygous F2 generation zebrafish, and positive homozygous zebrafish were independently selected from PCR and Sanger sequencing.

3. The construction method according to claim 2, characterized in that, The PCR method uses the detection primer pairs shown in SEQ ID NO.6~SEQ ID NO.

7.

4. A kit for constructing a zebrafish model of congenital pure red blood cell aplastic anemia based on bysl gene knockout, characterized in that, It includes an sgRNA that targets exon 1 of the zebrafish bysl gene; the targeting sequence of the sgRNA is shown in SEQ ID NO.1 and SEQ ID NO.2; it also includes the Cas9 protein; The zebrafish model of congenital pure red blood cell aplastic anemia based on bysl gene knockout was constructed using the method described in any one of claims 1 to 3.

5. The reagent kit according to claim 4, characterized in that, It also includes one or more of PCR reaction buffer and PCR reaction solution.

6. The reagent kit according to claim 5, characterized in that, The PCR reaction solution includes Taq DNA polymerase, dNTPs, and Mg. 2+ One or more of them.

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