A method for creating a gene deletion zebrafish line by knocking out ppia and application thereof

Abstract

The application discloses a method for creating a gene deletion zebrafish strain by knocking out ppia and application thereof, and belongs to the technical field of molecular biology and gene engineering. The application is based on CRISPR / Cas9 gene editing technology, a specific sgRNA target point is designed for a 2nd exon region of a zebrafish ppia gene, high-quality sgRNA is prepared by in-vitro transcription and is assembled with a recombinant Cas9 protein to form an RNP complex, and the RNP complex is microinjected into a single-cell-stage zebrafish embryo; a stable hereditary ppia gene frame shift mutation deletion strain is obtained through F0 generation chimera screening and backcrossing with a wild type. The application adopts an RNP injection strategy to avoid mRNA translation lag, improve editing efficiency and specificity, and reduce off-target risk through double screening of CHOPCHOP and CRISPRscan. The created strain completely destroys a CypA protein conservative functional domain, fills a blank of genetic tools for in-vivo function research of CypA of osteichthyes, and can be used for research of biological processes such as protein folding, immunoregulation and virus replication and related drug screening and evaluation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for creating gene-deleted zebrafish strains by knocking out ppia and its application. Background Technology

[0002] Cyclophilin A (CypA) is a typical peptidyl prolyl cis-trans isomerase encoded by the ppia (Peptidylprolylisomerase A) gene, widely distributed in various eukaryotic cells from yeast to humans. CypA not only participates in basic cellular physiological processes such as protein folding and transport, and signal transduction, but also plays a crucial role in inflammation regulation, immune responses, apoptosis, and the replication and infection cycle of various viruses (such as HIV and HCV). Due to the high conservation of its functional domains in vertebrates, CypA has become one of the important targets for immunology, virology, and anti-tumor drug development. In the field of functional genomics research, establishing stable knockout animal models of specific genes is the most direct and effective means of elucidating the biological functions of genes in vivo.

[0003] Currently, research on CypA function mainly relies on small interfering RNA knockdown in mammalian cell lines, in vitro protein purification experiments, or mouse gene knockout models. However, cellular knockdown struggles to simulate the complex microenvironment and multi-system interactions of the organism, and RNA interference suffers from limitations such as off-target effects and incomplete inhibition. While mouse models can reflect mammalian physiological characteristics, they have drawbacks such as long reproductive cycles, high feeding costs, and the opacity of embryos hindering real-time observation of developmental dynamics. Zebrafish, as the second largest vertebrate model organism after mice, shares 87% genetic homology with humans and possesses unique advantages such as embryonic transparency, in vitro development, short generation cycles, high reproductive capacity, and mature genetic manipulation capabilities, making it widely used in developmental biology, disease modeling, and drug screening. In recent years, although CRISPR / Cas9 technology has been widely applied to zebrafish gene editing, it remains a gap in the construction of specific knockout strains of the ppia gene in bony fishes. Existing technologies are mostly focused on other immune or metabolic-related genes and suffer from problems such as high sgRNA off-target rates, unstable editing efficiency, crude identification of F0 generation chimeras, and a lack of precise frameshift mutation screening. Furthermore, traditional Cas9 mRNA injection strategies suffer from drawbacks such as translational lag leading to misaligned splicing windows and the accumulation of translational products in vivo causing nonspecific toxicity. The lack of stable and reliable CypA-deficient zebrafish genetic tools limits in-depth analysis of CypA function within the evolutionary context of fish and vertebrates, and also hinders the full utilization of the high-throughput screening advantages of zebrafish models for drug evaluation of CypA-related pathways. Therefore, there is an urgent need to establish an efficient, specific, and stably heritable method for creating zebrafish ppia gene knockout strains to fill this technological gap. Summary of the Invention

[0004] To address the aforementioned problems, this invention first provides a method for creating gene-deleted zebrafish strains by knocking out ppia, comprising the following steps: designing at least one sgRNA target site targeting the coding sequence of the zebrafish ppia gene, wherein the target site is located within the second exon region and is adjacent to a PAM sequence with an NGG end; synthesizing a target-specific DNA oligonucleotide chain and annealing and extending it with Cas9 Scaffold Oligo to obtain a double-stranded DNA transcription template; performing in vitro transcription of sgRNA using T7 RNA polymerase and purifying it by ethanol precipitation; mixing recombinant Cas9 protein and purified sgRNA at a mass ratio of 2:1 and incubating to form an RNP complex; microinjecting the RNP complex into single-cell stage zebrafish fertilized eggs; cultivating F0 generation chimeric adult fish, backcrossing them with wild-type zebrafish to obtain F1 generation, and screening for zebrafish strains carrying frameshift mutations that delete the ppia gene through genotyping.

[0005] In some embodiments, the sgRNA target screening follows these conditions: the 5′ end of the target is preferably diguanine GG or monoguanine G; off-target prediction is performed using the CHOPCHOP tool and the off-target risk is marked in green or yellow; the prediction score using CRISPRscan is greater than 60 and the target avoids the presence of consecutive repeating bases.

[0006] In some embodiments, the sgRNA is purified by chemical precipitation using anhydrous ethanol and sodium acetate after in vitro transcription. The purified product has an A260 / A280 ratio between 1.8 and 2.0 as determined by NanoDrop, and the bands are confirmed to be single and clear by 1.5% agarose gel electrophoresis.

[0007] In some embodiments, the final concentration of Cas9 protein in the RNP complex is 300-500 ng / μL, the final concentration of sgRNA is 150-250 ng / μL, and 0.05% phenol red is added as a tracer at the final concentration during injection, with an injection volume of approximately 1 nL.

[0008] In some embodiments, the F0 generation editing activity screening is performed as follows: genomic DNA is extracted from embryos 24-48 hours after injection, PCR amplification and Sanger sequencing are performed using target-specific primers, and chimeric editing efficiency is evaluated by peak characteristics of the sequencing peak diagram.

[0009] In some embodiments, the F1 generation genotype identification uses the TA cloning precise identification method: the F1 generation PCR product is ligated with the pMD19-T vector, transformed into DH5α competent cells, positive single colonies are selected and verified by colony PCR using M13 universal primers, and then sent for testing. The wild-type sequence is compared using SnapGene software to screen for frameshift heterozygotes caused by non-3-fold base deletion.

[0010] In some embodiments, the zebrafish is an AB-type wild-type zebrafish, and the breeding conditions are a water temperature of 28.0℃, a pH of 6.5~7.5, and a 14h light / 10h dark cycle.

[0011] The present invention also provides a zebrafish strain with ppia gene deletion created by the above method, which is a stable genetic strain with loss of CypA function and carries an Indel frameshift mutation in the exon region of the ppia gene.

[0012] This invention also provides the application of the above-mentioned zebrafish strain with ppia gene deletion in the preparation of a CypA protein functional research model.

[0013] This invention also provides the application of the above-mentioned zebrafish strain with ppia gene deletion in the screening and evaluation of drugs related to protein folding and transport, immune regulation, inflammatory response, apoptosis or viral replication.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects: (1) In this invention, recombinant Cas9 protein and sgRNA are directly assembled into an RNP complex for microinjection, which avoids the lag in the in vivo translation of traditional Cas9 mRNA, significantly improves the instantaneous shearing efficiency in fertilized eggs, and reduces non-specific editing and off-target effects.

[0015] (2) This invention uses CHOPCHOP and CRISPRscan online tools to screen sgRNA targets, limiting the target to the first 2 / 3 of the CDS region of the ppia gene and the second exon, ensuring that the introduced Indel mutation causes a frameshift, completely destroying the conserved functional domain of the CypA protein and achieving complete loss of gene function.

[0016] (3) This invention uses TA cloning combined with Sanger sequencing to accurately identify the F1 generation genotype, accurately identify frameshift mutant individuals with non-triple base deletion, ensure the genetic stability and genotype accuracy of the obtained zebrafish strain, and facilitate long-term generation and phenotypic analysis.

[0017] (4) This invention establishes a stable knockout heterozygous strain of the ppia gene in zebrafish for the first time, providing an irreplaceable genetic model for in vivo functional studies of CypA in bony fish in the fields of protein transport, inflammation and immunity, apoptosis and pathogen infection, and can be applied to high-throughput drug screening and evaluation. Attached Figure Description

[0018] Figure 1 A schematic diagram of the target sequence and mRNA sequence for the ppia gene knockout in zebrafish. Figure 2This is a graph showing the results of primer genome-specific detection and analysis; Figure 3 Schematic diagram of primer sequence design for gRNA; Figure 4 This is an electrophoresis verification image of the sgRNA in vitro transcription template DNA; Figure 5 This is an image of purified sgRNA detected by agarose gel electrophoresis. Figure 6 A schematic diagram of a single-cell microinjection procedure in zebrafish. Figure 7 Agarose gel electrophoresis image of F0 generation PCR amplification products; Figure 8 This is a graph showing the results of Sanger sequencing peak analysis (overlapping peaks) near the target site in the F0 generation. Detailed Implementation

[0019] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments. Example

[0020] I. Experimental Materials The experimental animals used in this invention were healthy, wild-type (WT) AB zebrafish bred in the laboratory. The experimental fish were maintained daily in a flowing, independent zebrafish rearing system. The physical and chemical parameters of the water within the system were strictly controlled under standard rearing conditions: water temperature was maintained at 28.0℃, and pH was adjusted to between 6.5 and 7.5. The photoperiod was set to an automatic alternation of 14 hours of light and 10 hours of darkness to simulate the natural diurnal rhythm.

[0021] The main experimental instruments and equipment used in this invention include: a stereomicroscope manufactured by Nikon for embryo observation and micromanipulation; a micro-pneumatic syringe (specifically model PV830 or PV850) manufactured by WPI for high-precision control of injection volume; a micromanipulator manufactured by Sutter Instruments for precise needle positioning; a 5417R high-speed refrigerated centrifuge manufactured by Eppendorf for centrifugation during nucleic acid purification; a PCR amplification instrument and a 3730 series high-throughput Sanger sequencer manufactured by Applied Biosystems; and a gel imaging system manufactured by Bio-Rad for digital acquisition and analysis of electrophoretic bands.

[0022] The main core reagents and consumables used in the embodiments of this invention include: commercial recombinant Cas9 protein and high-fidelity PCR MasterMix enzyme produced by New England Biolabs (NEB); one-step sgRNA in vitro transcription kit produced by Beyotime Biotechnology Co., Ltd.; borosilicate glass capillaries (glass injection needles) for preparing microinjection needles produced by Sutter Instruments Co., Ltd.; TA cloning sequencing vector (pMD19-T vector) and DH5α competent cells produced by Takara Bio Inc.; and highly safe GelRed nucleic acid dye, high-precision DNA marker, and RNase-free water produced by Sangon Biotech (Shanghai) Co., Ltd.

[0023] II. Experimental Methods 1. gRNA primer design and target site screening Based on the mRNA and genomic sequence of the zebrafish ppia gene (searched from NCBI and Ensembl databases), the following principles were followed to design target sites for targeted knockout: The Cas9 target site must contain 20 bases, and in order to facilitate efficient in vitro transcription of T7 RNA polymerase, the 5′ end sequence of the target site is preferably diguanine (GG) or monoguanine (G); the three bases immediately adjacent to the 3′ end of the target site constitute the protospacer adjacent motif (PAM region), and the sequence is strictly required to be NGG.

[0024] The target site is selected in the first two-thirds of the gene coding sequence (CDS region) and located after the ATG start codon. This invention preferably locates the target site in the second exon of the ppia gene. This approach can completely disrupt the conserved functional domain of the CypA protein after introducing a frameshift mutation, achieving complete loss of function.

[0025] The exon sequences of the ppia gene were input into the online design tool CHOPCHOP, and preliminary screening was performed using zebrafish genome alignment and off-target prediction algorithms. Sequences with off-target risk were marked in green or yellow. Subsequently, candidate targets were submitted to the CRISPRscan website, which specializes in predicting editing efficiency in zebrafish, and sequences with a score greater than 60 and avoiding sequences containing consecutive repetitive bases (such as TTTT) were selected. Finally, three highly efficient and specific target sites were identified for subsequent experiments.

[0026] 2. In vitro transcription and chemical purification of sgRNA High-quality, high-purity sgRNA was prepared using a commercial one-step in vitro transcription system. The specific purification and operation steps are as follows: Transcription template preparation: The synthesized and PAGE-purified target-specific DNA oligonucleotide chain was mixed in an equimolar amount with the Cas9 Scaffold Oligo provided in the transcription kit in a centrifuge tube. 2× DNA polymerase premix was added, and RNase-free water was added to a total volume of 20 μL. Annealing and extension reactions were performed in a PCR instrument: denaturation was first performed at 95 °C for 2 min, followed by a very slow cooling rate of 0.1 °C / s to 25 °C, allowing the two oligonucleotide chains to perfectly complement each other and be extended by polymerase into a complete double-stranded DNA transcription template.

[0027] In vitro transcription reaction: In the reaction tube after the above extension is completed, add 2uL of 10× transcription buffer, 2uL of T7 RNA polymerase mixture and 2uL of NTP premix (25mM each), mix well and incubate at 37℃ for 3-4 hours. The sgRNA chain is synthesized by T7 RNA polymerase catalysis.

[0028] Removal of template DNA: After transcription is complete, add 1 μL of DNase I enzyme without RNase activity to the system and incubate at 37°C for 15-30 min to completely degrade the residual DNA template.

[0029] Chemical precipitation and purification: Nucleic acid purification was performed using the classic anhydrous ethanol and sodium acetate method. The reaction mixture was brought to a volume of 100 μL, followed by the addition of 10 μL of 3M sodium acetate (pH 5.2) and 250 μL of pre-chilled anhydrous ethanol. After gently mixing with a pipette tip, the mixture was placed in a -20°C freezer for overnight precipitation (or -80°C for 30 min). The centrifuge was pre-cooled to 4°C and centrifuged at 12,000 rpm for 15 min to allow the sgRNA to form a small precipitate. The supernatant was then carefully aspirated. The nucleic acid precipitate was washed with 200 μL of 75% pre-chilled ethanol prepared with RNase-free water, centrifuged at 4°C for 5 min, and the ethanol was discarded. The remaining trace liquid at the bottom of the tube was carefully aspirated again with a 10 μL pipette tip, and the tube was allowed to air dry at room temperature for 5–10 min. Finally, 30–50 μL of RNase-free water was added, and the precipitate was gently dissolved. Its absorbance and concentration were measured using a NanoDrop2000 micrometer (ensuring A...). 260 / A 280 The ratio is between 1.8 and 2.0. 2 μL of the product is added to Loading Buffer and verified by 1.5% agarose gel electrophoresis to check whether the bands are single, clear and undegraded.

[0030] 3. RNP complex preparation and single-cell microinjection RNP complex preparation: To avoid the lag in in vivo translation of recombinant mRNA and improve cleavage efficiency, this invention employs direct assembly of recombinant Cas9 protein and purified sgRNA. Commercial Cas9 protein and in vitro transcribed and purified sgRNA are mixed at a mass ratio of 2:1 in a sterile, enzyme-free centrifuge tube (in the final injection system, the final concentration of Cas9 protein is controlled at 300-500 ng / µL, and the final concentration of sgRNA is controlled at 150-250 ng / µL). The mixture is incubated at room temperature for 5 min to allow for complete binding and entanglement to form a functional ribonucleoprotein (RNP) complex. Before injection, a high-purity phenol red solution with a final concentration of 0.05% is added as a microinjection indicator, and the volume is made up with RNase-free water.

[0031] Microinjection Procedure: The night before injection, sexually mature, healthy adult fish were placed in a spawning tank with a partition at a female-to-male ratio of 1:2. The following morning, 1-3 hours after the lights were turned on, the partition was removed to stimulate natural mating and spawning, and the fertilized eggs were collected quickly. Under a microscope, fertilized eggs in the 1-cell stage (within 30 minutes of fertilization, before cell division) were neatly arranged in an agarose-coated culture dish. Using a micropneumatic syringe, a glass injection needle was precisely inserted into the yolk sac of the embryo, injecting approximately 1 nL of RNP complex solution using pneumatic pressure. Simultaneously, 15 fertilized eggs without any injection were set up as a normal control group. After injection, the embryos were carefully transferred to a culture dish containing standard E3 embryo culture medium and incubated in a 28.5°C incubator. The E3 culture medium was changed daily, and dead or malformed fertilized eggs were aspirated and removed. When the juvenile fish reach 5 dpf, they are fed high-density paramecium, and then gradually transitioned to live brine shrimp. They are raised under strict conventional and standardized conditions until sexual maturity, thus obtaining F0 generation chimeric adult fish.

[0032] 4. F0 generation selection, breeding, and F1 generation heterozygous line genotyping Rapid screening of F0 generation editing activity: 24–48 hours after the knockout microinjection, 15 embryos from the injection group were randomly collected and placed in 1.5 mL centrifuge tubes. 100 μL of tissue lysis buffer (containing 10 mmol / L Tris-HCl pH 8.0, 50 mmol / L KCl, 1.5 mmol / L MgCl2, 0.45% NP-40, 0.45% Tween-20, and 0.2 mg / mL proteinase K) was added, and the mixture was incubated at 55°C for 3 hours. Subsequently, the proteinase K was inactivated at 95°C for 10 min, and the supernatant was collected after centrifugation at maximum speed for 5 min to obtain the genomic DNA template for F0 generation individuals (or a crude extraction method using 0.1 M NaOH can be used). PCR amplification was performed using two pairs of specific identification primers designed for the knockout target site (Test-F1 / R1, expected product 340 bp; Test-F2 / R2, expected product 480 bp). PCR products were detected by 1.5% agarose gel electrophoresis (containing 0.5 μg / mL GelRed dye, electrophoresis at 120V constant voltage for 30 min). The presence of single-specific amplification bands of the expected size was observed using a gel imaging system. The remaining PCR amplification products were directly sent to a sequencing company for Sanger sequencing. The peak characteristics near the splicing sites were analyzed using sequencing peak diagrams to evaluate the chimeric editing efficiency of the F0 generation.

[0033] F1 generation heterozygous breeding and precise identification of TA cloning: Pre-numbered, sexually mature F0 generation chimeric adult fish, screened and confirmed to have a high efficiency mutation peak, were backcrossed with age-matched purebred wild-type (WT) zebrafish at a female-to-male ratio of 1:2. Fertilized eggs were collected the following day and incubated in E3 medium. When the F1 generation larvae reached 5 dpf, caudal fin tissue was excised from individual larvae or whole larvae (n=50) to extract their independent genomic DNA templates. PCR amplification was performed again using the aforementioned specific primer pairs. Since small Indel mutations (1-10 bp) cannot directly increase migration distance in agarose gel electrophoresis, the PCR products that showed normal electrophoresis were purified using a gel extraction and purification kit, and then efficiently ligated with the pMD19-T cloning vector at 16°C for 2 hours. The ligation product was chemically transformed into competent cells and evenly spread on LB agar plates containing 100 μg / mL ampicillin. The plates were then incubated overnight at 37°C. Ten white positive single colonies were randomly selected from each sample and inoculated into LB liquid medium. Colony PCR was performed using the M13 universal primers for verification. The amplified positive clones were sent for Sanger sequencing. The returned sequencing base sequences were locally aligned with the standard wild-type reference sequence of the zebrafish *ppia* gene using SnapGene software to accurately analyze the mutation type (deletion, insertion, or substitution number). Finally, F1 generation heterozygous individuals carrying non-3-fold base deletions that could cause a complete frameshift were selected and propagated to create homozygous mutant strains.

[0034] Experimental Results and Analysis 3.1 Target design, primer confirmation and specificity detection 3.1.1 Target Design and Screening To achieve efficient and precise editing of the ppia gene in zebrafish, this experiment first performed sequence retrieval and analysis of the target gene. Subsequently, the online design tool Cas-Designer was used to analyze the sequences, identifying three sgRNA targets with high predictive efficiency and low off-target risk, such as... Figure 1 As shown. They are all located in the coding sequence region of the zebrafish ppia gene (within the first three exons), and their sequences are as follows: sgRNA-1: 5′-AAGTGTTTTCGATCTGACC-3′ (PAM: NGG, SEQ ID No: 1); sgRNA-2: 5′-GGCTATGGCTACAAGGGATC-3′ (PAM: NGG, SEQ ID No: 2); sgRNA-3: 5′-GTCAATGGCCAATGCTGGCC-3′ (PAM: NGG, SEQ ID No: 3); 3.1.2 Primer specificity detection Analysis using an online specificity detection website showed that the three sets of primers exhibited good specificity across the entire genome, with no completely identical duplicate targets found. Figure 2 ).

[0035] 3.1.3 Primer Determination To obtain sgRNA for gene editing, DNA template primers were designed based on the three targets mentioned above. gRNA primer design is as follows: Figure 3 .

[0036] 3.2 sgRNA in vitro transcription and microinjection 3.2.1 In vitro transcription of sgRNA Before in vitro transcription, the template DNA was first amplified. Using the Cas9 vector sequence driven by the T7 promoter as a template, PCR amplification was performed using the primers described in section 4.1 above, followed by electrophoresis verification. The electrophoresis results showed that the DNA template was a single, clear band, without any diffuse extraneous bands, approximately 100 bp in size, indicating high purity and integrity of the template DNA, suitable for subsequent in vitro transcription reactions. Figure 4 ).

[0037] Three sgRNAs were synthesized using a one-step sgRNA in vitro transcription kit, each using an amplified DNA fragment as a template. The purified products were analyzed by 1.5% agarose gel electrophoresis. The results showed that each sgRNA presented as a single bright band at approximately 100 bp, without degradation tails or nonspecific bands. Figure 5 ).

[0038] 3.2.2 Microinjection Each sgRNA was separately mixed with Cas9 protein to form an RNP complex, which was then microinjected into zebrafish single-cell embryos. The experimental procedure was as follows: Figure 6 .

[0039] 3.3 F0 generation PCR detection and knockout efficiency analysis Based on the location of the knockout target site in the genome, two pairs of specific primers were designed to amplify the relevant DNA fragments for sequencing analysis of changes in the knockout target site. Two pairs of specific primers: Test-F1: 5′-GTCCACTGCGCCTTCATGAC-3′ (SEQ ID No: 4); Test-R1: 3′-GCTCTGCCGTATTGCAGCATG-5′ (SEQ ID No: 5); Test-F2: 5′-GTCCGAAAGGTTGAACTCGGT-3′ (SEQ ID No: 6); Test-R2: 3′-CCACCGTCTTCATACAGGCT-5′ (SEQ ID No: 7).

[0040] Twenty-four hours after the knockout procedure, 15 injected embryos were collected. Genomic DNA was crudely extracted using 0.1M NaOH and then used as a template for PCR amplification. The expected product lengths for the two primer pairs were 340 bp and 480 bp, respectively. Agarose gel electrophoresis results showed (e.g.) Figure 7 The sample showed a specific band consistent with the expected knockout fragment size. Sequencing analysis of the remaining PCR products revealed that both primer pairs showed double peaks (overlapping peaks) near the target site, indicating that the appendage sequence had mutated or recombinated, and the knockout was effective. Figure 8 ).

[0041] This study used zebrafish as a model organism and employed CRISPR / Cas9 gene editing technology to design three sgRNA targets in the ppia (CypA) gene. Through in vitro transcription to synthesize sgRNA, microinjection of the RNP complex, F0 generation screening, and sequencing verification, a stably heritable frameshift mutant heterozygous strain was successfully obtained. The following is a systematic discussion of this study from four aspects: technical verification, the significance of the preliminary results, comparison with previous studies, and limitations and future prospects.

[0042] At the technical validation level, sgRNA design in the CRISPR / Cas9 system is the primary step determining knockout efficiency and specificity. This study utilized the Cas-Designer website to rapidly and efficiently design CRISPR / Cas9 targets and corresponding primers. Optimizing the design principles of zebrafish sgRNA and analyzing its sequence characteristics significantly improved the system's cleavage efficiency. Analysis using an online specificity detection website showed that all three primer sets perfectly matched the target gene sequence, with no completely identical sites detected with other genes in the genome, meeting the specificity criteria for primer design. Electrophoresis of the in vitro transcribed template DNA confirmed a single, clear band without any diffuse extraneous bands, indicating high template purity and integrity. The three sgRNAs synthesized using a one-step sgRNA in vitro transcription kit all showed a single, bright band at approximately 100 nt on electrophoresis, without degradation tails or non-specific bands, indicating good transcription product quality.

[0043] In terms of genotype identification, the specific PCR primers designed in this study were used to amplify the genomic DNA of F0 generation individuals. The agarose gel electrophoresis results showed a single band, and sequencing analysis indicated the presence of a heterozygous deletion mutation.

[0044] The significance of these preliminary results is that this study is the first to establish a stable knockout heterozygous strain of the ppia gene in zebrafish, filling a gap in genetic tools for studying the in vivo function of CypA in bony fishes. Due to its advantages such as transparent embryos, mature genetic manipulation techniques, and high homology with human genes, zebrafish has become an indispensable vertebrate model for functional genomics research.

[0045] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for creating a gene-deleted zebrafish strain by knocking out ppia, characterized in that, Includes the following steps: At least one sgRNA target was designed targeting the coding sequence of the zebrafish ppia gene. The target was located in the second exon region and immediately adjacent to the 3′ end of the PAM sequence with NGG. A target-specific DNA oligonucleotide chain was synthesized and annealed and extended with Cas9 Scaffold Oligo to obtain a double-stranded DNA transcription template. The sgRNA was transcribed in vitro using T7 RNA polymerase and purified by ethanol precipitation. The recombinant Cas9 protein and purified sgRNA were mixed at a mass ratio of 2:1 and incubated to form an RNP complex. The RNP complex was microinjected into single-cell stage zebrafish zygotes. F0 generation chimeric adult fish were obtained by culturing and backcrossing with wild-type zebrafish to obtain F1 generation. Zebrafish strains carrying frameshift mutations and ppia gene deletion were screened by genotyping.

2. The method according to claim 1, characterized in that, The sgRNA target screening follows these conditions: the 5′ end of the target is preferably diguanine GG or monoguanine G; off-target prediction is performed using the CHOPCHOP tool and the off-target risk is marked in green or yellow; the prediction score using CRISPRscan is greater than 60 and the target should avoid containing consecutive repeating bases.

3. The method according to claim 1, characterized in that, The sgRNA was transcribed in vitro and then purified by chemical precipitation using anhydrous ethanol and sodium acetate. The purified product was detected by NanoDrop and the A260 / A280 ratio was between 1.8 and 2.

0. The bands were also confirmed to be single and clear by 1.5% agarose gel electrophoresis.

4. The method according to claim 1, characterized in that, The final concentration of Cas9 protein in the RNP complex is 300-500 ng / μL, the final concentration of sgRNA is 150-250 ng / μL, and 0.05% phenol red is added as a tracer at the final concentration during injection, with an injection volume of approximately 1 nL.

5. The method according to claim 1, characterized in that, The F0 generation editing activity screening process involves: collecting embryos 24-48 hours after injection to extract genomic DNA, performing PCR amplification and Sanger sequencing using target-specific primers, and evaluating chimeric editing efficiency by analyzing the peak characteristics of the sequencing peaks.

6. The method according to claim 1, characterized in that, The F1 generation genotype identification was performed using the TA cloning precise identification method: the F1 generation PCR product was ligated with the pMD19-T vector, transformed into DH5α competent cells, and positive single colonies were selected and verified by colony PCR using M13 universal primers before being sent for testing. The wild-type sequence was compared using SnapGene software to screen for frameshift heterozygotes caused by non-3-fold base deletion.

7. The method according to claim 1, characterized in that, The zebrafish in question are wild-type AB zebrafish, and the breeding conditions are water temperature 28.0℃, pH 6.5~7.5, and a 14h light / 10h dark cycle.

8. A zebrafish strain with a ppia gene deletion created by the method of any one of claims 1 to 7, which is a stable genetic strain with loss of CypA function and carries an Indel frameshift mutation in the exon region of the ppia gene.

9. The application of the zebrafish strain with the ppia gene deletion as described in claim 8 in the preparation of a CypA protein functional research model.

10. The application of the zebrafish strain with the ppia gene deletion as described in claim 8 in the screening and evaluation of drugs related to protein folding and transport, immune regulation, inflammatory response, apoptosis, or viral replication.

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