GRNA and application thereof in construction of new ricefield eel strain with high 1alpha, 25-dihydroxyvitamin D3 level

Abstract

The invention discloses gRNA (guide ribonucleic acid) and application thereof in construction of a new ricefield eel strain with a high 1alpha, 25-dihydroxy vitamin D3 level, belongs to the technical field of genetic engineering, and provides gRNA with a nucleotide sequence as shown in SEQ ID NO.1. The invention further discloses a preparation method of the gRNA. The gRNA is applied to construction of a new ricefield eel strain with a high 1alpha, 25-dihydroxy vitamin D3 level. The gRNA is applied to construction of a fast-growing disease-resistant new ricefield eel strain with the high 1alpha, 25-dihydroxy vitamin D3 level. A target spot of the cyp24a1 gene of the ricefield eel with high specificity is designed, gRNA is synthesized in vitro, the cyp24a1 gene capable of degrading the 1alpha, 25 (OH) 2D3 is quickly knocked out, a new ricefield eel strain with the high 1alpha, 25-dihydroxy vitamin D3 level is obtained, the 1alpha, 25 (OH) 2D3 has the functions of promoting fish growth and improving fish immunity, and the ricefield eel has important application value.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and in particular relates to gRNA and its application in constructing a new strain of yellow eel with high 1α,25-dihydroxyvitamin D3 level. Background Art

[0002] A deficiency of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in the human body not only affects growth and development, but also impairs immune function and inflammatory responses, and increases the risk of various diseases. Due to insufficient dietary 1α,25(OH)2D3 supply, 1α,25(OH)2D3 deficiency is now widespread.

[0003] Yellow eel (Swinhoe's alpaca) is a high-quality, specialty aquatic product. Its bones are used as medicine in traditional Chinese medicine, and its meat and blood also have medicinal value in traditional Chinese medicine, making it a popular choice among consumers. Growth traits are a key production trait of concern in the aquaculture industry. Recent research suggests that 1α,25(OH)2D3 deficiency can significantly impact fish growth and metabolism. Furthermore, infectious diseases caused by bacteria and viruses in farmed animals cause significant losses to the aquaculture industry and have become a bottleneck restricting its sustainable development. Recent research has also shown that supplementing feed with 1α,25(OH)2D3 can significantly enhance aquatic animals' resistance to viruses and bacteria. These results suggest that 1α,25(OH)2D3 may play a role in regulating growth and immune function in aquatic animals. Therefore, developing a new yellow eel strain with high 1α,25(OH)2D3 levels, rapid growth, and excellent disease resistance is of great significance to the industry's development. Summary of the Invention

[0004] To solve the above technical problems, the present invention proposes gRNA and its application in constructing a new strain of eel with high 1α,25-dihydroxyvitamin D3 levels. According to the principles of gene editing technology, a target site of the eel cyp24a1 gene with high specificity is designed, and gRNA is synthesized in vitro to quickly knock out the cyp24a1 gene that degrades 1α,25(OH)2D3, so that the eel body is rich in 1α,25(OH)2D3.

[0005] To achieve the above object, the present invention provides a gRNA, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] The present invention also provides the use of the gRNA in constructing a new strain of yellow eel with high 1α,25-dihydroxyvitamin D3 levels.

[0007] Preferably, the gRNA targets the cyp24a1 gene of the yellow eel, and the nucleotide sequence of the cyp24a1 gene is shown as SEQ ID NO.6.

[0008] Further preferably, the gRNA increases the level of 1α,25-dihydroxyvitamin D3 in the eel by targeted knockout of the cyp24a1 gene of the eel, thereby obtaining a new strain of eel with high 1α,25-dihydroxyvitamin D3 level.

[0009] The present invention also provides the use of the gRNA in constructing a new fast-growing and disease-resistant rice field eel strain with high 1α,25-dihydroxyvitamin D3 levels.

[0010] The present invention also provides specific primers for the gRNA, the nucleotide sequence of the upstream primer of the gRNA is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer of the gRNA is shown in SEQ ID NO.5.

[0011] The present invention also provides the use of the specific primers of the gRNA in constructing a new strain of yellow eel with high 1α,25-dihydroxyvitamin D3 levels. The specific primers of the gRNA improve the level of 1α,25-dihydroxyvitamin D3 in the yellow eel by targeted knockout of the yellow eel cyp24a1 gene, thereby obtaining a new strain of yellow eel with high 1α,25-dihydroxyvitamin D3 levels.

[0012] The present invention also provides a method for constructing a new strain of yellow eel with high 1α,25-dihydroxyvitamin D3 level, comprising the following steps:

[0013] (1) Design gRNA targeting the cyp24a1 gene based on CRISPR / Cas9 technology;

[0014] (2) constructing an expression vector containing the gRNA described in step (1), and performing in vitro transcription to obtain gRNA;

[0015] (3) injecting the gRNA and Case9 protein obtained in step (2) into a wild-type single-cell stage rice field eel embryo to obtain an injected rice field eel embryo;

[0016] (4) breeding the injected eel embryos obtained in step (3) into P0 generation eels, performing genotyping on the P0 generation eels, and screening for P0 generation mutant eels with cyp24a1 gene knockout in which the gRNA target sites on both the sense and antisense strands are mutated;

[0017] (5) selecting a female mutant from the P0 generation of mutant eels described in step (4) and mating with a wild-type male eel to perform genotyping, and screening an F1 generation of mutant female eels with cyp24a1 gene knockout in which the gRNA target sites on both the sense and antisense strands are mutated;

[0018] (6) Selecting male mutants from the P0 generation of mutant eels described in step (4) to mate with the F1 generation of mutant female eels described in step (5) to obtain F2 generation embryos, and raising them to sexual maturity, and screening to obtain F2 generation cyp24a1 gene knockout homozygous mutants, that is, obtaining a new strain of eels with high 1α,25-dihydroxyvitamin D3 levels.

[0019] Preferably, the nucleotide sequence of the gRNA in step (1) is as shown in SEQ ID NO.1.

[0020] Preferably, the nucleotide sequence of the cyp24a1 gene in step (1) is shown as SEQ ID NO.6.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects:

[0022] The principle of the present invention is that vitamin D3 (VD3) is a prohormone that is inactive by itself. It needs to be converted into 25(OH)D3 by the 25-hydroxylase encoded by the CYP2R1 gene in the liver, and then hydroxylated for the second time by the 1α-hydroxylase encoded by CYP27B1 to form biologically active 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). 1α,25(OH)2D3 and excess 25(OH)D3 are further metabolized by the 24-hydroxylase encoded by the cyp24a1 gene into inactive 23-hydroxylated products or 24-hydroxylated products (such as Figure 1 The cyp24a1 gene is located on chromosome 6 and has 12 exon regions. Knocking out the cyp24a1 gene in the yellow eel can block the hydroxylation of 25(OH)D3 and 1α,25(OH)2D3, maintaining high 1α,25(OH)2D3 levels in the fish.

[0023] Based on the principles of gene editing technology, this study designed a highly specific guide RNA targeting the cyp24a1 gene in the yellow eel. The guide RNA was synthesized in vitro, the Cas9 protein and guide RNA were thoroughly mixed, and the mixture was injected into wild-type yellow eel embryos at the one-cell stage. Sequencing was performed on newly emerged fry to verify the effectiveness of the target. Tail-chopping sequencing was performed on yellow eels of appropriate size to identify the yellow eel mutant P0. These mutants were then backcrossed with wild-type yellow eels to obtain the F1 generation, and heterozygous mutants from the F1 generation were identified. Male and female F1 heterozygous mutants were then self-crossed, and sequencing was performed to identify homozygous mutants from the F2 generation lacking the cyp24a1 gene. This construction method can rapidly knock out the cyp24a1 gene, which degrades 1α,25(OH)2D3, resulting in an enrichment of 1α,25(OH)2D3 in yellow eels. Since 1α,25(OH)2D3 has the function of promoting fish growth and enhancing fish immunity, this fish has important industrial application value.

[0024] The construction method of the present invention obtains a new strain of eel with high 1α,25-dihydroxyvitamin D3 level, providing people with a new dietary source of 1α,25(OH)2D3, which will help solve the increasingly serious problem of 1α,25(OH)2D3 deficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 It is the activation and metabolism process of vitamin D3;

[0027] Figure 2 Schematic diagram of the target gRNA for cyp24a1 gene knockout;

[0028] Figure 3 This is the result of gRNA effectiveness test for cyp24a1 gene target;

[0029] Figure 4 The P0 generation cyp24a1 gene knockout mutant rice field eel;

[0030] Figure 5 This is the effectiveness test result of gRNA targeting cyp24a1 gene in F1 generation;

[0031] Figure 6 This is an F2 generation cyp24a1 gene knockout homozygous mutant eel. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example 1

[0038] 1. Method for constructing a new strain of eel with high 1α,25-dihydroxyvitamin D3 levels

[0039] 1. Target design

[0040] According to the sequence of the cyp24a1 gene of the yellow eel (NCBI accession number NW_018127881.1, SEQ ID NO. 6), the target site of the cyp24a1 gene was designed according to the target site design principles through the CRISPR / Cas9 target site design website (https: / / www.crisprscan.org / sequence / ), and a site was selected in the 8th exon region on the antisense strand to obtain gRNA (such as Figure 2 shown).

[0041] The nucleotide sequence of gRNA is SEQ ID NO.1: TGTCCACACGGATCCTGGTCGGG.

[0042]

[0043] 2. Design of detection primers

[0044] Design target gRNA-specific PCR primers. Primers were designed using Primer 5.0 software based on the genomic sequence of the yellow eel cyp24a1 gene. The gRNA upstream primer, eel-cyp24a1-F: CCGATGTGCACCGTGTA (SEQ ID NO. 4), and the gRNA downstream primer, eel-cyp24a1-R: CTCAGGCTCTGTACTTTGC (SEQ ID NO. 5), were used to test the effectiveness of target knockout.

[0045] 3. gRNA DNA Template Design

[0046] The gRNA DNA template sequence consists of a T7 promoter sequence, a sequence encoding a target-specific gRNA, and a constant region of crRNA / tracrRNA. The gRNA is amplified by PCR using the pGH-T7-zCas9 plasmid as a template using primers with nucleotide sequences such as SEQ ID NO.2 and SEQ ID NO.3 to obtain an amplified product, which is then transcribed. The T7 promoter sequence is connected to the gRNA target sequence as a template and PCR amplified using T7 promoter universal primers. The amplification system is shown in Table 1, and the amplification procedure is shown in Table 2.

[0047] SEQ ID NO. 2: TAATACGACTCACTATAGTGTCCACACGGATCCT.

[0048] SEQ ID NO. 3: TTCTAGCCTAAAACACCAGGATCCGTGTGGACA.

[0049] Table 1 Amplification system

[0050] Components Dosage <![CDATA[Phusion TM High-Fidelity PCR Master Mix (2X)]]> 12.5μL Tracr fragment + T7 primer mix 1 μL 0.3 μM target F1 / R1 oligonucleotide mix 1 μL Enzyme-free water 10.5μL

[0051] Table 2 Amplification procedures

[0052]

[0053]

[0054] 4. Transcription to generate gRNA

[0055] The transcription system is shown in Table 3.

[0056] Table 3 Transcription system

[0057] reactants volume NTPmix 8μL gRNADNA template 6μL 5X transcription reaction buffer 4 μL Transcriptase cocktail 2μL

[0058] Prepare the system according to Table 3, mix thoroughly, centrifuge, and incubate at 37°C for 3 h. Immediately after the transcription reaction, add 1 μL of DNase and incubate at 37°C for 15 min.

[0059] 5. gRNA Purification

[0060] The in vitro transcribed gRNA was purified using a kit (purchased from Thermo Fisher, catalog number A29377). The purification procedure was as follows: first, enzyme-free water was added to make the total volume 200 μL. Then, 100 μL of binding buffer was added and pipetted to mix thoroughly. Then, 300 μL of anhydrous ethanol was added and pipetted to mix thoroughly. The mixture was then transferred to a GeneJET PCR agarose gel tube with a collection tube. TM Centrifuge the RNA purification column at room temperature and 14,000 rpm for 60 seconds, then discard the solution. Add 700 μL Wash Buffer 1 (make sure 13 mL of >96% anhydrous ethanol has been added for dilution), centrifuge at room temperature and 14,000 rpm for 60 seconds, then discard the solution. Add 700 μL Wash Buffer 2 (make sure 30 mL of >96% anhydrous ethanol has been added for dilution), centrifuge at room temperature and 14,000 rpm for 60 seconds, then discard the solution. Add 700 μL Wash Buffer 2 again, centrifuge at room temperature and 14,000 rpm for 60 seconds, then discard the solution. Centrifuge the empty tube at room temperature and 14,000 rpm for 60 seconds (the residual anhydrous ethanol in the RNA sample will inhibit the reaction of downstream enzymes). Then transfer the column to a new 1.5 mL In the EP tube, add 15 μL of enzyme-free water to the column, centrifuge at room temperature, 14000 rpm for 60 seconds; re-aspirate the centrifuged solution and add it to the tube in the column, centrifuge again at 14000 rpm for 60 seconds (to improve the recovery rate); finally, measure the concentration with a micro-spectrophotometer, store the original solution in a liquid nitrogen tank, and store the diluted solution at -80°C for later use.

[0061] 6. Injection of Cas9 protein and gRNA into eel embryos

[0062] The injection solution was prepared by mixing 1 μL of Cas9 protein (5000 ng / μL), 1 μL of gRNA (1000 ng / μL), 0.5 μL of phenol red solution (0.5%), and 2.5 μL of enzyme-free water. Microinjection instrument parameters were adjusted. Normal, wild-type, single-cell eel embryos were selected and neatly arranged on an injection mold. The mixture was microinjected into the embryo yolk at a volume of 10 nL per embryo. The injected embryos were immediately placed in aerated water for 24 hours and incubated at 28°C.

[0063] 7. Cyp24a1 gene target site effectiveness detection

[0064] After embryos reached 5–6 cm in size, sections of the tail fin were excised after anesthesia and placed in PCR tubes. Lysis buffer (20 μL) was added and the cells were lysed at 60°C for 30 min to obtain DNA. Amplification was performed using the following PCR reaction system: 12.5 μL of 2× PCR Taq Mix; 1 μL of each 10 μM upstream and downstream primers; 2 μL of 100 ng / μL genomic DNA; and 8.5 μL of ddH₂O. The amplification protocol was as follows: initial denaturation at 94°C for 10 min; 39 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s; followed by annealing at 72°C for 10 s.

[0065] The amplified PCR products were sequenced (e.g. Figure 3 As shown), determine whether the target site is mutated based on the peak graph. If overlapping peaks appear and the overlapping peak positions are consistent with the expected ones, it is the mutant P0 generation (as shown in Figure 4 shown).

[0066] 8. Screening of F1 mutants of the cyp24a1 gene in the yellow eel

[0067] Since the first sexual maturity of eels is female, the P0 generation female mutants were mated with wild-type male eels. After the offspring were raised to a suitable size, they were anesthetized, tail-clipped, and the genome of the offspring was extracted according to the above method. After PCR amplification, the genome was sequenced. If the sequencing results showed a double peak at the target position, it was the F1 generation eel with the cyp24a1 gene deleted (such as Figure 5 and raised to sexual maturity.

[0068] 9. Screening of homozygous mutants of the F2 generation of the cyp24a1 gene in the yellow eel

[0069] The first sexually mature F1 generation eels are all female. At this time, the P0 generation eels are just reversed to male. The F1 generation female and P0 generation male eels with the same type of cyp24a1 gene knockout are screened and self-fertilized to obtain F2 generation embryos. They are raised to sexual maturity and the F2 generation cyp24a1 gene knockout homozygous mutants are screened (such as Figure 6 As shown in the figure), a new strain of eel with high 1α,25(OH)2D3 level and cyp24a1 gene knockout was successfully obtained.

[0070] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A gRNA, characterized in that The nucleotide sequence of the gRNA is shown in SEQ ID NO.

1.

2. Use of the gRNA as described in claim 1 in constructing a new strain of eel with high 1α,25-dihydroxyvitamin D3 levels.

3. The application according to claim 2, characterized in that: The gRNA targets the cyp24a1 gene of the yellow eel, and the nucleotide sequence of the cyp24a1 gene is shown in SEQ ID NO.

6.

4. The application according to claim 3, characterized in that The gRNA targets and knocks out the cyp24a1 gene of the rice field eel, thereby increasing the level of 1α, 25-dihydroxyvitamin D3 in the rice field eel and obtaining a new rice field eel strain with high 1α, 25-dihydroxyvitamin D3 levels.

5. Use of the gRNA as described in claim 1 in constructing a new strain of fast-growing and disease-resistant eels with high 1α,25-dihydroxyvitamin D3 levels.

6. The gRNA-specific primer according to claim 1, wherein The nucleotide sequence of the upstream primer of the gRNA is shown in SEQ ID NO.4, and the nucleotide sequence of the downstream primer of the gRNA is shown in SEQ ID NO.

5.

7. The use of the gRNA-specific primers according to claim 6 in constructing a new strain of eel with high 1α,25-dihydroxyvitamin D3 levels, characterized in that: The specific primers of the gRNA target and knock out the cyp24a1 gene of the rice field eel, thereby increasing the level of 1α, 25-dihydroxyvitamin D3 in the rice field eel and obtaining a new rice field eel strain with high 1α, 25-dihydroxyvitamin D3 level.

8. A method for constructing a new strain of eel with high 1α,25-dihydroxyvitamin D3 level, characterized in that: The following steps are involved: (1) Design gRNA targeting the cyp24a1 gene based on CRISPR / Cas9 technology; (2) constructing an expression vector containing the gRNA described in step (1), and performing in vitro transcription to obtain gRNA; (3) injecting the gRNA and Case9 protein obtained in step (2) into a wild-type single-cell stage rice field eel embryo to obtain an injected rice field eel embryo; (4) breeding the injected eel embryos obtained in step (3) into P0 generation eels, performing genotyping on the P0 generation eels, and screening for P0 generation mutant eels with cyp24a1 gene knockout in which the gRNA target sites on both the sense and antisense strands are mutated; (5) selecting a female mutant from the P0 generation of mutant eels described in step (4) and mating with a wild-type male eel to perform genotyping, and screening an F1 generation of mutant female eels with cyp24a1 gene knockout in which the gRNA target sites on both the sense and antisense strands are mutated; (6) Selecting male mutants from the P0 generation of mutant eels described in step (4) to mate with the F1 generation of mutant female eels described in step (5) to obtain F2 generation embryos, and raising them to sexual maturity, and screening to obtain F2 generation cyp24a1 gene knockout homozygous mutants, that is, obtaining a new strain of eels with high 1α,25-dihydroxyvitamin D3 levels.

9. The method for constructing a new strain of eel with high 1α,25-dihydroxyvitamin D3 level according to claim 8, characterized in that: The nucleotide sequence of the gRNA in step (1) is shown in SEQ ID NO.

1.

10. The method for constructing a new strain of rice field eel with high 1α,25-dihydroxyvitamin D3 level according to claim 8, characterized in that: The nucleotide sequence of the cyp24a1 gene in step (1) is shown in SEQ ID NO.6.

Images