LPLa gene deleted zebrafish primer and preparation method of zebrafish

Abstract

The invention discloses an LPLa gene deleted zebra fish primer and a preparation method of zebra fish, belongs to the technical field of molecular biology, and particularly relates to design and application of the LPLa gene deleted zebra fish primer. The LPLa gene deleted zebrafish primer disclosed by the invention consists of a forward primer and a reverse primer of two sgRNA (small guide ribonucleic acid); the method comprises the following steps: 1, synthesizing two sgRNA chains; 2, combining the two sgRNA chains with Cas9 protein, and carrying out micro-injection on the combined sgRNA chains and Cas9 protein into fertilized eggs in a single cell period of zebra fish; 3, detecting sgRNA knockout efficiency, and sequencing and screening mutant zebrafish F0; 4, backcrossing to obtain a mutant BC1 generation, and sequencing to detect the frame shift condition after the gene is knocked out; and 5, selfing to obtain F1-generation zebrafish LPLa- / -, and subsequently culturing to obtain the LPL gene deletion homozygous mutation line. The primer disclosed by the invention realizes accurate and efficient knockout of a target gene in a zebrafish genome, and the embryo development after microinjection does not have malformation, so that the LPLa gene deleted zebrafish mutant strain is obtained. The LPLa gene deletion mutant zebrafish provides a basic model for fish fat development and deposition regulation.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to the design and application of primers for zebrafish with LPLa gene deletion. Background Technology

[0002] Aquatic products, as an important source of animal protein, have significant advantages such as safe meat quality, abundant yield, diverse varieties, and the absence of zoonotic diseases. Their muscle tissue is rich in various essential amino acids, occupying an irreplaceable position in the national dietary structure. Currently, the evaluation of aquatic product muscle quality mainly draws on the testing standards for livestock and poultry meat. Its nutritional components primarily include protein, fat, moisture, ash, and fatty acids. The most significant difference in the conventional nutritional components of fish lies in their fat content. Intramuscular fat is mainly divided into intramuscular fat and intermuscular fat. Intramuscular fat contains triglycerides and phospholipids; triglycerides are believed to play a regulatory role in fat deposition. Phospholipids contain various unsaturated fatty acids beneficial to the human body. Among the fatty acid components of phospholipids, unsaturated fatty acids such as linoleic acid, oleic acid, and palmitoleic acid are positively correlated with meat flavor, while saturated fatty acids such as palmitic acid, myristic acid, and stearic acid are negatively correlated with meat flavor. When intramuscular fat contains more triglycerides and phospholipids, and a higher content of unsaturated fatty acids, the fish meat quality is superior to that of meat with low intramuscular fat content, and its appearance is also more lustrous. Therefore, intramuscular fat is considered a key indicator for meat quality grading. .

[0003] Multiple studies have shown that the PPAR signaling pathway is an important pathway for regulating the differential deposition of intramuscular and peritoneal fat in animals such as pigs, cattle, and chickens. LPL is a key factor in lipolysis within this pathway. PPARγ, PPARα, and glucose can increase the expression level of LPL gene mRNA in macrophages; PPARα and AMPK affect the activity of LPL in cardiomyocytes; the use of PPARα activators reduces the activity of LPL in cardiomyocytes; and during fasting, the expression of PPARγ and cholesterol regulatory element-binding protein 1 is reduced in adipose tissue. LPL is a key enzyme that plays a limiting role in lipolysis. LPL is synthesized by the parenchymal cells of organisms (skeletal muscle cells, adipocytes, macrophages, etc.) and then infiltrates into the bloodstream. LPL not only hydrolyzes protein-associated triglycerides but also participates in the metabolic pathways of phospholipids in the body. Polyploidy exists in fish genes. In the red sea bream (Pagrus major) Redfin pufferfish (Takifugu rubripes) and Clark's hooked trout (Oncorhynchus clarkii) Medaka (Oryzias latipes) Two LPL homologs (LPLa and LPLb) were discovered in polyploid fish. The expression level of LPLa in tissues such as liver, fat, and muscle was more than 10 times that of LPLb. This indicates that homologous genes have different expression characteristics during evolution. In carp with high intramuscular fat content, LPLa was found to be significantly overexpressed in muscle but not significantly changed in liver (Zhang et al., 2024). Differential metabolites were also enriched in the glycerol lipid metabolism pathway where LPLa is located. The results further demonstrate that LPLa may be involved in the regulation of high intramuscular fat deposition in carp. The LPLa knockout zebrafish model will provide an important model for the study of fat deposition regulation in fish.

[0004] The application and development of CRISPR / Cas9 gene editing technology has provided a revolutionary tool for research in functional genomics and molecular mechanisms. It can simultaneously target and modify single or multiple target genes. The high efficiency, flexibility, and precision of CRISPR / Cas9 technology greatly improve the accuracy and efficiency of constructing corresponding gene deletion biological models. Zebrafish, as a model organism, has had its fundamental molecular mechanisms thoroughly studied. Its transparent embryos and rapid growth and development provide a more direct way to observe the impact of gene deletions on early development and to more quickly construct homozygous lines with gene deletions. (Invention Content)

[0005] This invention provides an LPLa gene-deleted zebrafish primer and a method for preparing the primer in zebrafish. The LPLa gene-deleted zebrafish primer of this invention consists of two sgRNA primers: a forward primer and a reverse primer. The forward primer sequences are: sgRNA 1, TTCTAATACGACTCACTATA GCTACGATCTGGAGAACCAC GTTTTAGAGCTAGA; sgRNA 2, TTCTAATACGACTCACTATA GGTTGGAAGGTCCCACCATT GTTTTAGAGCTAGA; and the reverse primer sequence is: 5'-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'.

[0006] The method for preparing LPLa gene-deleted zebrafish using the primers described in this invention is carried out according to the following steps:

[0007] I. Two sgRNA chains, sgRNA 1 and sgRNA 2, were synthesized in vitro using the above-mentioned LPLa gene-deleted zebrafish primers.

[0008] 2. Two sgRNA chains were bound to Cas9 protein and microinjected into zebrafish single-cell stage fertilized eggs.

[0009] 3. The efficiency of sgRNA knockout was tested by collecting fertilized eggs injected 48 h after fertilization. The mutant zebrafish were fed to age of more than 1 month and frameshifted zebrafish F0 were selected by sequencing.

[0010] IV. The mutant F0 generation zebrafish were backcrossed with wild type to obtain the mutant BC1 generation. This generation of zebrafish was raised to more than 1 month old, and the frameshift after the knockout of this gene was detected by sequencing.

[0011] 5. Select zebrafish with the same mutation and frameshift position for self-crossing. Select mutants with identical frameshift positions after gene knockout and self-crossing to obtain F1 generation zebrafish LPLa. - / - Subsequently, a line was established and cultured to obtain a homozygous mutant line with the LPL gene deletion.

[0012] Furthermore, the method for synthesizing the sgRNA chain in vitro in step one:

[0013] I. Obtaining LPL gene deletion zebrafish primers: Design core target primers, selecting two target sites in exon 5 with a distance of no more than 80 bp as core target sequences: LPL, 1 5'-GCTACGATCTGGAGAACCACGGG-3', LPL, 2 5'-GGTTGGAAGGTCCCACCATTGGG-3'; Based on the two core target sites, obtain LPL gene deletion zebrafish primers, where the forward primer sequence for sgRNA 1 is: 5'-TTCTAATACGACTCACTATA GCTACGATCTGGAGAACCACGTTTTAGAGCTAGA-3'; the forward primer sequence for sgRNA 2 is: 5'-TTCTAATACGACTCACTATAGGTTGGAAGGTCCCACCATT GTTTTAGAGCTAGA-3'; the reverse primer sequences for both sgRNA 1 and sgRNA 2 are:

[0014] 5'-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3';

[0015] 2. In step one, the two forward primers are combined with the reverse primers respectively to obtain the sgRNA in vitro transcription template by PCR; two sgRNA strands are synthesized by in vitro transcription using an sgRNA synthesis kit at 37°C for 6 h.

[0016] Furthermore, in step one, the forward primer consists of a T7 promoter sequence, a modified core target sequence, and an sgRNA scaffold. The T7 promoter sequence is: 5'-TTCTAATACGACTCACTATA-3'; the sgRNA scaffold is: 5'-GTTTTAGAGCTAGA-3'.

[0017] Furthermore, the core target sequence modification method is as follows: the 5' end of the core target sequence should be a G base, eliminating the "NGG" base in the target PAM region; the addition of a G base at the 5' end of the core target is not a requirement of the Cas9 target itself, but because the sgRNA in vitro transcription vector used adopts the T7 promoter, which requires the first two positions of the transcription start site to be GG; the three bases immediately adjacent to the 3' end of the target constitute the protospacer adjacent motif (PAM) region, which is required to be "NGG", where N is any base.

[0018] Furthermore, in step two, the concentration ratio of the two sgRNAs is 1:1.

[0019] Furthermore, in step two, the microinjection system consists of 2 μL of Cas9 protein, 0.8 μL of 10×NEBuffer, 1600 ng of sgRNA, and ddH2O to a final volume of 6 μL.

[0020] Furthermore, the method for verifying the sgRNA knockout efficiency in step three is as follows: genomic DNA is extracted from fertilized eggs, gene fragments are amplified by PCR, and sgRNA knockout efficiency is verified by polypropylene gel electrophoresis. The detection primers are: LPL-E5-SF 5'-TCACTTCTTCAATCTTCAGGCA-3'; LPL-E5 SR 5'-ACCAGTAGTGGCCACCATTAAC-3'.

[0021] Furthermore, in step four, when backcrossing with wild-type zebrafish, select zebrafish of a different sex from the original fish for one-to-one backcrossing.

[0022] The beneficial effects and advantages of this invention are as follows:

[0023] 1. The primers described in this invention are designed by selecting a suitable target site sequence at exon 5 of the zebrafish LPL gene and designing LPL gene deletion probe primers. The primers of this invention can achieve precise knockout of the target gene in the zebrafish genome with a knockout rate of 85%, and no malformation occurred in the embryonic development after microinjection.

[0024] 2. This invention utilizes a 1:1 mixture of two designed sgRNA strands targeting different sites, which are then injected into a single zebrafish zygote after binding to Cas9 protein. This allows for simultaneous knockout of two sites on the target gene, resulting in high knockout efficiency, a simple process, low gene mutation costs, and ease of widespread adoption. This invention also utilizes CRISPR / Cas9 technology to obtain an LPLa gene deletion mutant in zebrafish and establishes a stably heritable mutant strain.

[0025] 3. The LPLA constructed in this invention - / - The zebrafish mutant line with a 16 bp deletion predicts a significant change in protein structure compared to the wild type. The LPL gene deletion mutant zebrafish constructed in this invention can be used for research on lipid metabolism and deposition, providing a new technological approach for the breeding of high-quality aquatic products.

[0026] This invention selected a suitable target sequence in exon 5 of the zebrafish LPLa gene. The designed primers enabled precise and efficient knockout of the target gene in the zebrafish genome. No malformations were observed in the embryos after microinjection, successfully obtaining an LPLa gene deletion mutant strain. The obtained LPLa gene deletion mutant zebrafish provides a basic model for the regulation of fat development and deposition in fish.

[0027] The LPLa gene deletion primers described in this invention were used to prepare an LPLa gene-deleted zebrafish using CRISPR / Cas9 gene editing technology. Changes in key lipid parameters of the zebrafish were then detected, and the LPLa gene was constructed. - / - The zebrafish homozygous line provides a basic research platform for further exploring the specific physiological function of this gene in lipid metabolism of economic fish, and provides new technical means for the breeding of high-quality aquatic products. Attached Figure Description

[0028] Figure 1 The amplified band is sgRNA.

[0029] Figure 2 For the detection of mutation rate in LPL gene knockout F0 eggs;

[0030] Figure 3 The mutation status of the LPL gene knockout F0 generation up to 2 months of age;

[0031] Figure 4 Sequencing alignment diagram of the F1 generation of LPL gene knockout mutant;

[0032] Figure 5 Wild type (A) and LPLa - / - (B) 3D diagram of predicted structure of zebrafish LPL protein;

[0033] Figure 6 The relative expression levels of the LPL gene in zebrafish liver and muscle;

[0034] Figure 7 The content of T-CHO in the muscle of two zebrafish genotypes;

[0035] Figure 8 The TG content in the muscle of two zebrafish genotypes is shown. Detailed Implementation

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0038] Example 1: Design of the LPLa gene primers described in this invention (zebrafish LPLa gene deletion primers)

[0039] Specifically, this includes the design of the core target sequence and the synthesis of CRISPR probes. The forward primer consists of the T7 promoter sequence, the modified core target sequence, and the sgRNA scaffold, while the reverse primer uses the universal pT7 scaffold. The combination of the forward and reverse primers amplifies and replicates the sgRNA in vitro transcription template containing the T7 transcription promoter and the core target sequence. The T7 promoter sequence is 5'-TTCTAATACGACTCACTATA-3'; the sgRNA scaffold is 5'-GTTTTAGAGCTAGA-3'. The genomic data of the target gene LPLa was downloaded from the Zebrafish Genome Database (https: / / grch37.ensembl.org / index.html), and the core target primer was designed online for the CDS region using the CHOPCHOP website (https: / / chopchop.cbu.uib.no / ), selecting a suitable target sequence. In this embodiment, two target sites were ultimately selected, located on the negative strand of exon 5 of the same exon: LPL,1 5'-GCTACGATCTGGAGAACCACGGG-3', LPL, 2 5'-GGTTGGAAGGTCCCACCATTGGG-3'.

[0040] In this embodiment, the forward primer sequence of the final zebrafish LPLa gene deletion primer sgRNA chain is as follows: sgRNA1, TTCTAATACGACTCACTATA GCTACGATCTGGAGAACCAC GTTTTAGAGCTAGA; sgRNA2, TTCTAATACGACTCACTATA GGTTGGAAGGTCCCACCATT GTTTTAGAGCTAGA; reverse primer (universal pT7scaffold): 5'-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'.

[0041] Example 2: Preparation method of LPL gene deletion mutant zebrafish

[0042] This embodiment describes the preparation method and application of the zebrafish LPL gene deletion primers from Example 1 for constructing LPL gene deletion mutant zebrafish. The preparation method and application specifically include the following steps:

[0043] I. Using primers from zebrafish with the LPLa gene deletion described above, two sgRNA chains were synthesized in vitro, i.e., sgRNA chains were synthesized in vitro based on the target sequence:

[0044] The two sgRNA oligo forward primers from Example 1 above were combined with the universal reverse primer pT7scaffold to obtain sgRNA in vitro transcription templates via PCR. The PCR system for the sgRNA in vitro transcription templates is shown in Table 1. The PCR products were recovered using a purification and recovery kit (Gene JET Gel Extraction Kit, K0692), and the DNA quality and concentration of the products were determined. 300 ng of the PCR product was subjected to agarose gel electrophoresis, and the fragment size was detected to be approximately 120 bp. Figure 1 The amplified band is sgRNA, which is approximately 120 bp in length, and the marker is DL500.

[0045] sgRNA was synthesized in vitro by incubation at 37°C for 6 h using the HiScribe™ T7 Quick High Yield RNA Synthesis Kit (E2050S) (Table 2 sgRNA Synthesis). Residual DNA was removed, and the synthesized sgRNA was recovered using an RNA purification kit (MACHEREY-NAGEL). After determining its concentration and quality, 2 μL was aliquoted and stored at -80°C for later use.

[0046] Table 1 sgRNA PCR amplification system

[0047]

[0048] Table 2 sgRNA Synthesis

[0049]

[0050] 2. Two sgRNA chains were bound to Cas9 protein and microinjected into zebrafish single-cell stage fertilized eggs; the two sgRNAs were mixed at a concentration ratio of 1:1 and bound to Cas9 protein (NEB), and then microinjected into single-cell stage zebrafish embryos.

[0051] III. Detection of probe knockout efficiency in F0 generation zebrafish: Fertilized eggs injected 48 hours post-fertilization were used to test sgRNA knockout efficiency. F0 generation zebrafish were raised to over one month of age, and frameshift zebrafish were selected by sequencing. Specifically, 20 eggs injected at 48 hours post-fertilization (48 hpf) and one wild-type egg were randomly selected. Genomic DNA was extracted, gene fragments were amplified by PCR, and sgRNA knockout efficiency was tested by polypropylene gel electrophoresis. The detection primers were: LPL-E5-SF 5'-TCACTTCTTCAATCTTCAGGCA-3'; LPL-E5 SR 5'-ACCAGTAGTGGCCACCATTAAC-3'. Figure 2 The results of this embodiment for detecting the mutation rate of LPL gene knockout F0 eggs are as follows: Figure 2 As shown, the mutation rate was approximately 85% (17 / 20).

[0052] After the remaining zebrafish eggs were reared for 60 days, genomic DNA was extracted from the tails of the fish to examine for mutations, resulting in LPL gene-deleted F0 generation zebrafish. In this example, a total of 54 LPL gene knockout embryos survived and developed into individuals. PCR and polypropylene gel electrophoresis showed that the LPL gene target mutation rate in the surviving individuals was 77.78% (42 / 54). Some of the test results in this example are shown below. Figure 3 As shown, zebrafish individuals with a large deletion mutation in the F0 generation of LPL gene knockout were selected for subsequent breeding.

[0053] Fourth, select F0 generation zebrafish with mutations and backcross them with wild-type zebrafish to obtain mutant generation 0 (BC1 generation). Raise these zebrafish to more than 1 month old and sequence them to detect the frameshift after the knockout of this gene. Specifically, select 10 F0 generation zebrafish with obvious mutations and backcross them with wild-type zebrafish to obtain mutant generation 0 (BC1 generation). Raise these zebrafish to more than 1 month old and sequence them to screen for frameshift zebrafish for subsequent culture.

[0054] 5. Select zebrafish with the same mutation and frameshift position for self-crossing. Select mutants with identical frameshift positions after gene knockout and self-crossing to obtain F1 generation zebrafish LPLa. - / - .

[0055] LPLa - / - F1 generation zebrafish: female and male LPLa from the BC1 generation with frameshift mutations and consistent knockout locations. + / -The mutant heterozygous zebrafish were self-crossed to obtain F1 generation zebrafish. Theoretically, the F1 generation should contain three gene types: wild type, heterozygous mutant, and homozygous mutant. In the 43 selected embryos, the mutation rate test results showed that the ratio of wild type: heterozygous: homozygous was 10:23:10. The mutation ratio of LPL gene in the F1 generation basically conforms to Mendel's laws.

[0056] Figure 4 Sequencing alignment of the F1 generation of LPL gene knockout mutants (WT is wild type). The sequencing results show that the LPL knockout individuals have 2 and 14 bp less at target sites 1 and 2 respectively compared to the wild type, with a total deletion of 16 bp that is not an integer multiple of 3, thus causing a frameshift mutation in the gene. Figure 4 ).

[0057] Figure 5 Wild type (A) and LPLa - / - (B) 3D diagram of zebrafish LPL protein structure prediction. The wild-type zebrafish LPL gene encodes a 511 aa amino acid chain. Figure 5 A), the protein sequence is: MMFNKGRVSSAFLISWMYFVYISSGMETTIDPTAESITLSDIIGNATEWMMDFTDIESKFSFRTLEEPEDDLCYIVPGQPQSIKDCNFNTETKTFIVIHGWTVTGMFESWVPKLVTALYER EPSANVIVVDWLSRAQQHYPTSASYTKLVGKDVAKFVNWLQAEIDYPWEKLHLLGYSLGAHVAGIAGLLTKHKVNRITGMDPAGPTFEYADSLSTLSPDDANFVDVLHTNTRGSPDRSIGIQRPVGHIDI YPNGGTFQPGCDLQNTMLMVATTGLRNMDQIVKCSHERSIHLFIDSLVNQDHESMAFRCSSRDSFNKGMCLSCRKNRCNKVGYAVNKIRTRRSSKMYMKTREMMPYKVFHYQVKVHFFGKTQLSYTDQPM KISLYGIHGEKENIPYIMPTLNTNSTVSFLLTTDADIGELLMVKLLWEKDTLISWPWWNSDTFHIRKLRIKSGETQSKIIFSAKESEFSYLSRGGEAAVFVKDKEAQSSRKNQRLHKMKMHGSSFKQNTE;

[0058] The frameshift mutation is predicted to prematurely terminate the translation of this gene, encoding a 237-aa amino acid chain. Figure 5B), the protein sequence is: MMFNKGRVSSAFLISWMYFVYISSGMETTIDPTAESITLSDIIGNATEWMMDFTDIESKFSFRTLEEPEDDLCYIVPGQPQSIKDCNFNTETKTFIVIHGWTVTGMFESWVPKL VTALYEREPSANVIVVDWLSRAQQHYPTSASYTKLVGKDVAKFVNWLQAEIDYPWEKLHLLGYSLGAHVAGIAGLLTKHKVNRITGMDPAGPTFEYADSLSTLSPDDANFVDVLHTNTRFSRS.

[0059] Subsequent line establishment and culture yielded a homozygous mutant line with LPL gene deletion.

[0060] VI. LPLa - / - After zebrafish grew to adulthood, the expression levels of the LPL gene in their liver and muscle were examined to determine the differences compared to wild-type zebrafish. Figure 6 The relative expression levels of the LPL gene in zebrafish liver and muscle (AB represents wild type, *indicates significant difference P<0.05), such as Figure 6 As shown, the expression level of the LPL gene in LPLa - / - The amount of LPLa in zebrafish muscle tissue was significantly reduced compared to that in wild-type zebrafish muscle tissue. This method successfully constructed LPLa. - / - Zebrafish mutant strain.

[0061] VII. Regarding LPLa - / - The total cholesterol (T-CHO, Nanjing Jiancheng) and total triglyceride (TG, Nanjing Jiancheng) contents in the muscle of mutant zebrafish and wild-type zebrafish were measured. The TG content in the muscle of the gene-deleted zebrafish was significantly higher than that in the wild-type zebrafish (P<0.05). Figure 7 The content of T-CHO in the muscle of two zebrafish genotypes (WT was wild type, *indicates significant difference P<0.05); Figure 8 The TG content in the muscle of zebrafish of two genotypes (WT was wild type, and the difference in ns was not significant, P>0.05) was calculated. In this example, the T-CHO and TG contents in the muscle were 0.022 and 0.439 mmol / gprot, respectively. Knocking out the LPLa gene significantly reduced the T-CHO content in zebrafish muscle, but had no significant effect on the TG content. This indicates that knocking out the LPLa gene affects fat deposition in zebrafish, and the model was successfully constructed.

Claims

1. A zebrafish primer with LPLa gene deletion, characterized in that, A primer for zebrafish with LPLa gene deletion consists of two forward and reverse primers for sgRNAs. The forward primer sequence for sgRNA 1 is: 5'-TTCTAATACGACTCACTATA GCTACGATCTGGAGAACCAC GTTTTAGAGCTAGA-3'; the forward primer sequence for sgRNA 2 is: TTCTAATACGACTCACTATA GGTTGGAAGGTCCCACCATT GTTTTAGAGCTAGA; and the reverse primer sequences for both sgRNA 1 and sgRNA 2 are: 5'-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'.

2. A method for preparing LPLa gene-deleted zebrafish using the primers described in claim 1, characterized in that, The method for preparing LPLa gene-deleted zebrafish is carried out according to the following steps: I. Two sgRNA strands were synthesized in vitro using the LPLa gene-deleted zebrafish primers as described in claim 1.

2. Two sgRNA chains were bound to Cas9 protein and microinjected into zebrafish single-cell stage fertilized eggs.

3. The efficiency of sgRNA knockout was tested by collecting fertilized eggs injected 48 h after fertilization. The mutant zebrafish were fed to age of more than 1 month and frameshifted zebrafish F0 were selected by sequencing. IV. The mutant F0 generation zebrafish were backcrossed with wild type to obtain the mutant BC1 generation. This generation of zebrafish was raised to more than 1 month old, and the frameshift after the knockout of this gene was detected by sequencing.

5. Select zebrafish with the same mutation and frameshift position for self-crossing. Select mutants with identical frameshift positions after gene knockout and self-crossing to obtain F1 generation zebrafish LPLa. - / - Subsequently, a line was established and cultured to obtain a homozygous mutant line with the LPL gene deletion.

3. The method for preparing LPLa gene-deleted zebrafish according to claim 2, characterized in that, The method for synthesizing sgRNA chains in vitro in step one: I. Obtaining LPL gene deletion zebrafish primers: Design core target primers, selecting two target sites in exon 5 with a distance of no more than 80 bp as core target sequences: LPL 1 5'-GCTACGATCTGGAGAACCACGGG-3', LPL 2 5'-GGTTGGAAGGTCCCACCATTGGG-3'; Based on the two core target sites, obtain LPL gene deletion zebrafish primers, where the forward primer sequence for sgRNA 1 is: 5'-TTCTAATACGACTCACTATA GCTACGATCTGGAGAACCACGTTTTAGAGCTAGA-3'; the forward primer sequence for sgRNA 2 is: 5'-TTCTAATACGACTCACTATAGGTTGGAAGGTCCCACCATT GTTTTAGAGCTAGA-3'; the reverse primer sequences for both sgRNA 1 and sgRNA 2 are: 5'-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'; 2. In step one, the two forward primers are combined with the reverse primers respectively to obtain the sgRNA in vitro transcription template by PCR; two sgRNA strands are synthesized by in vitro transcription using an sgRNA synthesis kit at 37°C for 6 h.

4. The method for preparing LPLa gene-deleted zebrafish according to claim 3, characterized in that, In step one, the forward primer consists of the T7 promoter sequence, the modified core target sequence, and the sgRNA scaffold.

5. The method for preparing LPLa gene-deleted zebrafish according to claim 4, characterized in that, The T7 promoter sequence is: 5'-TTCTAATACGACTCACTATA-3'; the sgRNA scaffold sequence is: 5'-GTTTTAGAGCTAGA-3'.

6. The method for preparing LPLa gene-deleted zebrafish according to claim 2, characterized in that, In step two, the concentration ratio of the two sgRNAs is 1:

1.

7. The method for preparing LPLa gene-deleted zebrafish according to claim 2, characterized in that, In step two, the microinjection system consisted of 2 μL of Cas9 protein, 0.8 μL of 10×NEBuffer, 1600 ng of sgRNA, and ddH2O to a final volume of 6 μL.

8. The method for preparing LPLa gene-deleted zebrafish according to claim 2, characterized in that, The method for verifying sgRNA knockout efficiency in step three is as follows: Genomic DNA is extracted from fertilized eggs, gene fragments are amplified by PCR, and sgRNA knockout efficiency is verified by polypropylene gel electrophoresis. The detection primers are: LPL-E5-SF 5'-TCACTTCTTCAATCTTCAGGCA-3'; LPL-E5 SR5'-ACCAGTAGTGGCCACCATTAAC-3'.

9. The method for preparing LPLa gene-deleted zebrafish according to claim 2, characterized in that, In step four, when backcrossing with wild-type zebrafish, select zebrafish of a different sex from your own for a one-to-one backcross.

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