Seawater fish gene editing and mutant screening method

Abstract

The invention belongs to the technical field of cell biology, and particularly discloses a marine fish gene editing and mutant screening method which comprises the steps of sgRNA synthesis, Cas9-sgRNA RNP complex preparation, microinjection and mutant screening. The marine fish microinjection needle and the biological glue are used, so that the problem that microinjection of marine fish fertilized eggs is difficult is solved; on the basis, the mutants can be screened through PCR amplification, HRMA analysis and PAGE electrophoresis, so that the screening cost is greatly reduced; in addition, the mutant screening time is shortened from several weeks to several months to about four days, and an efficient and economical novel method is provided for marine fish gene editing and mutant screening.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell biology, in particular to a method for gene editing and mutant screening of seawater fish. BACKGROUND

[0002] At present, gene editing technology has certain application in fish, mainly including CRISPR / Cas9 technology, transcription activator-like effector nuclease (TALEN) technology and zinc finger nuclease (ZFN) technology, etc., among which, the CRISPR / Cas9 technology is one of the most widely used gene editing technologies at present. The fish genome can be modified at a fixed point by using gene editing technology to achieve the purpose of genetic function research, genetic improvement, etc. However, the research on gene editing technology of seawater fish is relatively less, and the reported technologies are as follows: the use of CRISPR / Cas9 technology in Pseudosciaena crocea improves the mutation rate of exogenous target genes and the hatching rate of fertilized eggs; the use of TALEN technology establishes the genome editing technology of seawater flatfish, and successfully knocks out the male gene dmrt1 of Cynoglossus semilaevis, proving that the dmrt1 gene is the male determining gene of Cynoglossus semilaevis; the ZFN technology can be used for gene editing of seawater fish, but due to its relatively complex design and construction, the requirement for technical personnel is high, and its application in seawater fish is temporarily not available. At present, the main mutant screening technologies are as follows: screening technology based on fluorescent marker genes; gene chip technology screening; high-throughput sequencing technology and cell line screening, etc., but the above mutant screening technologies consume relatively more time and cost, and cannot achieve low-cost, rapid detection.

[0003] In summary, the research on gene editing and mutant screening technology of seawater fish in the prior art is relatively less, and the existing technology has the following problems: first, the existing gene editing and mutant screening technology has high cost, and a large number of consumables and instrument equipment need to be purchased; and the operation is complex, and the technical level and experimental conditions of the researchers are required to be high; and the design process is time-consuming and laborious, and is limited in large-scale use and promotion. SUMMARY

[0004] In order to solve the above problems, the present application develops a low-cost, high-efficiency and rapid detection and screening technology suitable for gene editing and mutant screening of seawater fish, which can effectively reduce the cost, has the advantage of fast detection speed, and is expected to break through the existing difficulties and promote the efficient development of gene editing and mutant screening of seawater fish.

[0005] In order to achieve the above purpose, the specific technical scheme adopted by the present application is as follows:

[0006] A method for gene editing and mutant screening of seawater fish, comprising the following steps:

[0007] S1. Synthesis of sgRNA: design and synthesize sgRNA targeting the target gene of marine fish;

[0008] S2. Preparation of Cas9-sgRNA RNP complex: incubate Cas9 protein with sgRNA to form ribonucleoprotein (RNP) complex;

[0009] S3. Microinjection: drop biological glue on the fertilized egg, and after standing, inject Cas9-sgRNA RNP complex solution into the fertilized egg using a microinjection needle;

[0010] S4. Screening of mutants: after the fertilized eggs develop into embryos, embryos containing mutant characteristics are picked for genomic DNA extraction, and the extracted DNA is subjected to PCR and high-resolution melting curve analysis (HRMA) to detect mutations, and polyacrylamide gel electrophoresis (PAGE) is used for mutant verification. Specifically, HRMA analysis mainly screens by comparing the shapes of melting curves of different samples. Typically, homozygous samples have relatively sharp, symmetric single peaks, and are relatively fixed. Mutant samples have relatively smooth melting peaks, which are relatively wide, asymmetric or double-peak, and the melting peaks are more diverse, with individual differences. PAGE verification is determined by electrophoresis bands. Homozygous samples show a single electrophoresis band, and mutant samples show multiple bands.

[0011] Further, in step S1, the method for designing sgRNA is as follows:

[0012] a. Determine the target sequence: select a DNA sequence starting with GG, containing 18 nucleotides in the middle, and ending with nGG (n represents any base) on the same exon from the CDS region of the target gene sequence, and screen for a sequence with good specificity by comparing with the marine fish genome to serve as the target sequence;

[0013] b. Design sgRNA: according to the determined target sequence, extract its reverse complementary sequence, and add the sgRNA backbone sequence to the 3' end of the reverse complementary sequence to obtain sgRNA.

[0014] Further, in step S1, the method for synthesizing sgRNA is as follows:

[0015] a. Synthesis of recombinant plasmid: use pDR274 as the template plasmid for synthesizing sgRNA, perform plasmid cleavage, sgRNA primer annealing to form DNA double strands, and perform ligation reaction of the cleaved plasmid and the DNA double strands to construct the recombinant plasmid pDR274-sgRNA;

[0016] b. In vitro transcription: the recombinant plasmid is transformed into DH5a, and positive clones are screened; the correctness of sgRNA sequence in the recombinant plasmid is identified by colony PCR; the recombinant plasmid is extracted and linearized; and then the linearized plasmid is used as a template to transcribe sgRNA and purify it, obtaining sgRNA that can be used for CRISPR-Cas9 gene editing.

[0017] Further, in step S1, when the marine fish is large yellow croaker, the target sequence is 5'-GGT ATAT CCT GGT GCG GGG ACA GG-3'(SEQ NO: 1); the sgRNA sequence is 5'-GGU AUA UCC UGG UGC GGG ACA GGG UUU UAG AGC UAG AAA UAG CAA GUU AAA AU AAGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC UUU UAA A-3'(SEQ NO: 2); and the sgRNA primer includes a positive strand with the sequence of 5'-TAG GTAT ATC CT GGT GCG GGA C-3'(SEQ NO: 3) and a negative strand with the sequence of 5'-AAA CGT CCC GCA CCA GGA TAT A-3'(SEQ NO: 4).

[0018] Further, in step S2, the ratio of Cas9 protein to sgRNA is 1:(1-5), and after mixing, they are incubated at room temperature for 15 minutes.

[0019] Further, in step S3, the biological glue is prepared by the following method: methyl cellulose with a viscosity of 4000 cps is added to 1 / 4 of the total water amount of 70°C ultrapure water and dispersed while stirring with a magnetic stirrer to obtain a hot gel, and then the remaining 3 / 4 of 4°C ultrapure water is slowly added and stirred on ice to obtain the required biological glue.

[0020] Further, in step S3, the fertilized eggs are placed in the grooves of the agarose gel, and then biological glue is added to the fertilized eggs. After completion, it is left to stand for five minutes, and then microinjection is performed.

[0021] Further, in step S3, the microinjection needle is a glass needle with a needle tip length of 9 mm, a needle tip opening inner diameter of 0.5 μm, and a polished smooth surface.

[0022] Further, in step S4, the genomic DNA extraction method is: first, the embryos containing mutant characterization are ground; then, NaOH solution is added for alkaline lysis; and finally, centrifugation is performed to obtain genomic DNA.

[0023] Further, in step S4, the PCR reaction program is: 95.0 DEG C denaturation 3 minutes; 95.0 DEG C denaturation 15 seconds; 60.0 DEG C annealing 20 seconds; 72.0 DEG C extension 20 seconds; cycle to the second step, repeat 39 times; 95.0 DEG C denaturation 10 seconds; 65.0 DEG C to 95.0 DEG C melting curve analysis, the heating rate 0.2 DEG C / s, plate read is carried out.

[0024] Compared with the prior art, the present application has the following beneficial effects:

[0025] (1) Application of seawater fish microinjection needle and biological glue

[0026] The seawater fish microinjection needle and biological glue are used innovatively to solve the problems of hard eggshell and needle sticking during microinjection of seawater fish zygotes, so that the needle sticking problem is basically solved, and the speed and efficiency of microinjection are greatly improved.

[0027] (2) Simple and efficient variant screening method

[0028] The technical scheme of the present application has the advantages of low cost, time saving and high efficiency in screening seawater fish gene editing mutants, and has obvious advantages in the following two aspects:

[0029] ① Fast mutant screening speed. The present application uses alkaline lysis to lyse cells and release genomic DNA, then performs PCR, and then uses HRMA for preliminary screening, and then uses PAGE gel for secondary screening according to the results. Through the above steps, the time required for mutant screening is greatly shortened.

[0030] ② The technical cost of the present application is low. Only through the processes of PCR, HRMA, PAGE and other processes, the mutant can be screened, the reagents and consumables consumed are relatively less, and the cost of mutant screening is greatly reduced.

[0031] (3) Wide applicability of the technical scheme

[0032] The technical scheme of the present application has been successfully tested in Pseudosciaena crocea, and can be applied to different types of seawater fish. Through the present application, efficient and rapid gene editing and gene editing in seawater fish can be realized, the experimental cost is reduced, and the time required for the experiment is shortened, and the present application has a wide application prospect in seawater fish.

[0033] In summary, the technical scheme of the present application can reduce the cost of seawater fish gene editing and mutant screening technology and accelerate the detection speed. In terms of gene editing, the operation process and reagent use are optimized, the human, material and time costs are reduced, and the risk of off-target effect is reduced. In terms of mutant screening, the detection steps are simplified, the detection efficiency is improved, and accurate screening results can be obtained in a short time. Compared with general mutant screening technology, the mutant screening time of the present application is shortened from several weeks to several months to about four days. The present application provides a new efficient and economical method for seawater fish gene editing and mutant screening. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 : HRMA result diagram example in Example 1. In the figure: the black curve is the wild type homozygote, and the red curve is the mutant.

[0035] Figure 2 : PAGE result diagram example in Example 1. In the figure: lane 5 is the wild homozygote band, lanes 1 and 13 are DNA markers, and the remaining lanes are mutant individual bands.

[0036] Figure 3 : Abnormal otolith embryos observed under a body microscope in Example 2.

[0037] Figure 4 : Melting curve characteristics in Example 2. In the figure: the red curve is the wild type, and the green curve is the F0 generation otol1 gene knockout group.

[0038] Figure 5 : DNA PAGE gel electrophoresis result in Example 2. In the figure: lanes 1-4 are mutant individual bands, and the WT marker lane is the wild type homozygote band. DETAILED DESCRIPTION

[0039] The present application will be further described below in combination with the drawings and specific examples. In the following examples, the reagents involved are commercially available conventional reagents unless otherwise specified, and the experimental operations involved are conventional operations in the art unless otherwise specified.

[0040] Example 1: Seawater fish gene editing and mutant screening method

[0041] The present embodiment provides a seawater fish gene editing and mutant screening method, comprising the following steps:

[0042] 1. Synthesis of sgRNA

[0043] (1). Design sgRNA sequence

[0044] ①. Identify the target sequence. To determine the target sequence to be edited, it is first necessary to identify the sequence of the desired target gene in the genome. This sequence can be obtained by searching the NCBI database, and then screening from the CDS region for target sequences located on the same exon. The target sequence is a DNA sequence that begins with GG, contains 18 nucleotides in the middle, and ends with nGG (N represents any base).

[0045] ②. Design sgRNA. Based on the determined target sequence, extract its inverse complementary sequence. Add the sgRNA backbone sequence to the 3' end of the inverse complementary sequence. The sgRNA backbone sequence of the SpCas9 system used in this invention is “5'-GUU UUA GAGCUA GAAAUAGCA AGU UAA AAU AAG GCU AGU CCG UUA UCA ACU UGAAAAAGU GGC ACC GAGUCG GUG CUU UUA AA-3”.

[0046] ③. Specificity Assessment. To prevent off-target effects, i.e., the Cas9-sgRNA complex being cleaved at non-target sites in the genome, the specificity of the designed sgRNA needs to be assessed. Bioinformatics tools can be used to compare the designed sgRNA sequence with the entire genome sequence of marine fish to screen for highly specific sgRNAs. Typically, BLAST is performed in NCBI for specificity screening. By comparing the target sequence with the genome, it is determined that the designed sgRNA has no fragments with a similarity greater than 5% at other locations in the genome. The sgRNA will only bind to the target gene and exert its function, with a very low probability of off-target binding to other gene sites, indicating high specificity.

[0047] (2) Synthesis of sgRNA

[0048] ①. Strains culture and plasmid extraction:

[0049] E. coli DH5α was cultured to the logarithmic growth phase, and then subjected to cold shock (placement on ice) and heat shock (brief heating at 42°C) to make the cell membrane more permeable, facilitating the uptake of exogenous DNA, thus obtaining competent cells. The prepared competent cells were mixed with plasmid pDR274 (a commercial plasmid containing the T7 RNA polymerase promoter), and then placed on ice for a period of time to allow the plasmid DNA to enter the cells, which is transformation. The transformed cells were then allowed to recover at 37°C for a period of time to restore normal growth. The recovered cells were plated on solid medium containing antibiotics (such as kanamycin), and transformed colonies containing the plasmid were screened.

[0050] Reinoculate into LB liquid medium containing kanamycin, 37°C, 200 rpm culture overnight. Use TIANGEN's TIAnypearls Plasmid Extraction Mini Kit to extract plasmid pDR274, get high-purity plasmid, absorbance ratio A260 / A280 is 1.80-1.85, concentration ≥100 μg / mL.

[0051] 2. Cutting of plasmid pDR274:

[0052] Mix plasmid pDR274 with BsaI restriction endonuclease in appropriate buffer. Incubate at 37°C for 1-2 hours to allow BsaI to recognize and cut specific sites in plasmid pDR274. Take 1 μg of plasmid pDR274, add appropriate amount of CutSmart Buffer and BsaI enzyme, and add sterile water to a total volume of 10 μL. Incubate at 37°C for 1 hour to allow plasmid pDR274 to be cut at specific sites to form linear plasmid, facilitating subsequent ligation reaction.

[0053] 3. Primer annealing:

[0054] Dilute the positive and negative strands of the designed sgRNA primer to a concentration of 200 ng / μL. Take 5 μL of each of the diluted primer positive and negative strands, add 10 μL of sterile water (PCR water) to mix, so that the total volume is 15 μL and the concentration of the primer positive and negative strands is 40 μM. Place the mixture in a PCR instrument, anneal at 95°C for 5 min, then slowly cool to room temperature to form double-stranded DNA fragments.

[0055] 4. Ligation reaction:

[0056] Take an appropriate amount (2 μL) of cut plasmid pDR274 linear plasmid, add 5 μL of annealed double-stranded DNA fragments (the molar ratio of primer-ligation vector is 3:1). Add 1 μL of T4 DNA ligase and 2x Reaction Buffer, and make up the total volume to 10 μL with sterile water. Place the centrifuge tube in a 16°C water bath for incubation for 18 hours to allow the double-stranded DNA fragments to be connected with the linear plasmid to form the recombinant plasmid pDR274-sgRNA. After incubation, transfer the tube to -20°C for storage until use.

[0057] 5. Transformation and selection:

[0058] Take an appropriate amount of ligation product recombinant granules pDR274-sgRNA, add to E. coli DH5a competent cells, ice bath for 30 min, then 42°C heat shock for 90 s, then ice bath for 2 min. Add LB medium, 37°C, 200 rpm shaking culture for 1 h, so that the plasmid in the bacterial solution expresses antibiotic resistance genes. Coated on LB agar plates containing kanamycin, 37°C inverted culture overnight, screened for positive clones containing recombinant plasmid pDR274-sgRNA.

[0059] 6. Colony PCR identification:

[0060] Pick a single colony on the plate and inoculate in LB liquid medium containing kanamycin for further culture, 37°C, 200 rpm shaking culture for 3-5 h. Take an appropriate amount of bacterial solution, extract plasmid according to TIANGEN TIAnypearls Plasmid Extraction Mini Kit instructions. PCR verification using specific primers to confirm the correctness of the sgRNA sequence in the recombinant plasmid.

[0061] 7. Plasmid recovery and PCR amplification:

[0062] TIANprep Rapid Mini plasmid extraction kit was used to extract plasmid from liquid bacterial culture. According to the instructions of the kit, the steps including centrifugation, lysis, neutralization, adsorption, washing and elution were carried out, and the recombinant pDR274 plasmid was purified and recovered. In the PCR reaction system, appropriate primers and Q5 Hot Start high-fidelity DNA polymerase were added, and the recovered recombinant pDR274 was used as template for PCR amplification to achieve linearization. The PCR reaction system contains recombinant plasmid template, primer, dNTPs, Taq polymerase and buffer, etc. The amplification conditions are: denaturation at 94°C for 3 min; 25-30 cycles, each cycle including denaturation at 94°C for 30 s, annealing at 55-60°C for 30 s, extension at 72°C for 1-2 min; finally extension at 72°C for 5-10 min.

[0063] 7. sgRNA in vitro transcription:

[0064] After confirming the success of linearization by agarose gel electrophoresis, the PCR product is used as a template for in vitro transcription using the MEGAscript T7 Transcription Kit. In the in vitro transcription reaction system, ATP, CTP, GTP, UTP, reaction buffer, linear template DNA and enzyme mixture are added, and the operation is carried out according to the kit instructions. Generally incubate at 37°C for 1-2 hours to allow T7 RNA polymerase to catalyze the transcription of sgRNA. After the reaction is completed, the transcription product is purified using the MEGAshortscript T7 Transcription Kit, DNase I (RNase-free), DNase I (RNase-free) Stop Solution and 75% ethanol according to the kit instructions to remove unreacted nucleotides, primers and other impurities. Finally, the sgRNA is further purified by ethanol precipitation to obtain sgRNA that can be used for CRISPR-Cas9 gene editing.

[0065] 2. Mix Cas9 protein and sgRNA

[0066] The Cas9 protein used is a commercial nuclear localization protein (D0513S, Cas9 NLS (SpCas9-NLS), Biyun Tian). In the commercial Cas9 protein buffer, mix the Cas9 protein with the sgRNA to form a ribonucleoprotein (RNP) complex. The volume ratio of Cas9 protein to sgRNA is usually between 1:1 and 1:5, but the specific ratio may vary depending on the experimental target and cell type, and can be optimized according to experimental needs. After gently mixing, incubate at room temperature to allow the Cas9 protein to fully bind to the sgRNA. Before microinjection, dilute and process the mixed Cas9-sgRNA RNP complex to the concentration required for microinjection.

[0067] 3. Microinjection

[0068] (1). Prepare the microinjection needle for microinjection, the whole length of the needle is 90.00 mm, the outer diameter is 1.00 mm, the inner diameter is 0.60 mm, the needle tip length is 9 mm, the needle tip opening inner diameter is 0.5 μm, and the glass needle is polished smooth; prepare the biological glue, add the methyl cellulose with a viscosity of 4000 cps to 1 / 4 of the total water volume of ultrapure water (70°C) to disperse, and at the same time, use a magnetic stirrer to stir, obtain a hot gel, then slowly add the remaining 3 / 4 of the ultrapure water (4°C), and stir on ice to obtain the biological glue with a required concentration of 1%-5% (w / v); prepare a 2% agarose gel, weigh 0.8 g of agar powder in a conical flask, pour 40 mL of 1 × TAE solution, place it in a microwave oven to heat to melt, pour it into a clean culture dish with 20 mL of agarose solution, and place it at room temperature to wait for solidification, then place the embryo microinjection mold with the groove side facing the already solidified agarose gel, pour the remaining agarose solution in the bottle onto the culture dish from one side, cover the mold, and place it at room temperature to wait for solidification, then remove the mold, and the microinjection plate is ready.

[0069] (2). Use a 10 μL pipette to aspirate the mixed Cas9-sgRNA NPs injection solution, and slowly inject it into the injection needle.

[0070] (3). Use a rubber suction tube to select good quality zygotes under a body microscope and suck them out with the suction tube, and arrange them one by one in the agarose gel groove in the injection plate, the purpose is to facilitate fixation and rapid injection of fish eggs, then suck out the excess water. Then use a suction tube to aspirate the prepared biological glue and drop it on the arranged zygotes, after completion, stand for five minutes (so that the biological glue can fix the zygotes well in the groove), and then proceed to microinjection.

[0071] (4). Install the injection needle on the mechanical arm of the microinjection operator, adjust the microoperator under the body microscope, observe the position of the needle tip, and ensure that the needle tip is aligned with the zygote. Then gently pierce the zygote membrane, inject the injection solution under the fish yolk membrane near the nucleus, and after injection, withdraw the microinjection needle, and proceed to the next zygote injection.

[0072] 4. Mutant screening

[0073] (1) Genomic extraction

[0074] ①. Pick the 48-hour post-fertilization embryos containing mutant characteristics:

[0075] Use a clean suction tube to pick the embryos containing mutant characteristics under a body microscope.

[0076] ②. Grind the tissue:

[0077] Put the embryo into a sterile mortar, add an appropriate amount of sterile sand (or use a tissue grinder), and grind the tissue into a fine homogenate with a pestle until the cells are broken and the genomic DNA is released.

[0078] ③. Add NaOH solution:

[0079] Add 40 μL of 50 mM NaOH solution to the ground tissue homogenate, mix gently by vortexing to ensure that the NaOH fully lyses the cells and releases the genomic DNA. Incubate the mixture in a water bath at 95°C for 13 minutes, gently vortexing every few minutes to ensure complete cell lysis. Immediately after incubation, place the mixture on ice to cool and prevent excessive DNA degradation.

[0080] ④. Centrifugation and neutralization:

[0081] Place the cooled mixture in a tabletop centrifuge and centrifuge at 12000-14000 rpm for 3-5 minutes to precipitate the cell debris and impurities at the bottom. Carefully aspirate the supernatant (about 30-35 μL) and transfer it to a new sterile centrifuge tube. Add 4 μL of 1 M Tris-HCl solution (pH 8.0-8.5) and mix gently by vortexing to neutralize the pH of the solution and stabilize the DNA. Store the treated DNA solution at 4°C until further use.

[0082] (2). Perform PCR and high-resolution melting curve analysis (HRMA)

[0083] PCR system (1 sample): PCR water 2.6 μL, DNA solution 5 μL, forward primer 0.2 μL, reverse primer 0.2 μL, template / sample 2 μL, total volume 10 μL.

[0084] PCR program: denaturation at 95.0°C for 3 minutes; denaturation at 95.0°C for 15 seconds; annealing at 60.0°C for 20 seconds; extension at 72.0°C for 20 seconds; repeat step 2 for 39 times; denaturation at 95.0°C for 10 seconds; melting curve analysis from 65.0°C to 95.0°C at a rate of 0.2°C / s, and perform plate read.

[0085] The length of the PCR primer should not exceed 150 base pairs, and the optimal length of the product should be less than 100 base pairs.

[0086] Then prepare HRMA: After the PCR reaction is completed, do not open the PCR tube, and directly perform HRMA. Ensure that the PCR instrument is equipped with high-resolution melting curve analysis function and has installed the corresponding software for data acquisition and analysis.

[0087] Running HRMA: Start the HRMA program according to the operation manual of the PCR instrument. Perform melting curve analysis at a temperature range of 65.0-95.0°C with a temperature ramping rate of 0.2°C / s, while performing plate read to monitor the melting of DNA double strands in real time.

[0088] Data analysis: After HRMA is completed, analyze the melting curve data using the accompanying software. By comparing the melting curve shapes and melting temperatures Tm of different samples (mainly according to the melting curve shapes), it can be quickly detected whether there are mutations or genotype differences in the genomic DNA. Under normal circumstances, the melting curve of the wild-type sample will show a specific shape and Tm, while the sample containing mutations will show different melting curve characteristics.

[0089] The melting curve is shown in Figure 1 , the black curve is the wild-type homozygote, and the red curve is the mutant. The results of individuals with gene mutations will show two peaks and the peak value is lower than that of homozygous individuals, while homozygous individuals are single-peak.

[0090] (3). Polyacrylamide gel electrophoresis (PAGE) verification

[0091] Configure the PAGE gel, load the prepared DNA samples (PCR amplification products) into the gel wells, and ensure that the loading amount of each sample is moderate to avoid overloading. Place the gel in the electrophoresis tank, add electrophoresis buffer (such as 1×TBE buffer), and ensure that the gel is completely immersed in the buffer. Turn on the power supply and set the appropriate voltage and time for electrophoresis. For polyacrylamide gel electrophoresis, a constant voltage (such as 80-150V) is usually used, and the electrophoresis time is determined according to the thickness of the gel and the size of the sample, generally 30 minutes to 2 hours. After electrophoresis is completed, remove the gel from the electrophoresis tank, and use an appropriate staining agent (such as gelred staining) to stain the DNA, and then observe and analyze the electrophoresis results under a gel imaging system.

[0092] The electrophoresis result is shown in Figure 2 , the wild-type homozygote shows only one band (lane 5), which represents that the target gene of the individual's two homologous chromosomes is "completely unedited", the DNA sequence is uniform, the fragment length is consistent after PCR amplification, and only one band is shown in electrophoresis; the mutant shows multiple bands, because the first generation of knockout individuals are chimeras, and the mutation types of the same target in different cells are different, so multiple DNA band fragments of different lengths will be produced after PCR amplification, and multiple bands will appear in electrophoresis; lanes 1 and 13 are DNA markers.

[0093] Note: Figures 1-2For the results of zebrafish cyp19a1a mutation analysis, only as an example of the images of the two parts of HRMA and electrophoresis in this embodiment, the performance and feasibility of using HRMA and DNA PAGE for mutant screening are embodied, and the specific application of the whole method system in marine fish is embodied in embodiment 2.

[0094] Example 2: Application of marine fish gene editing and mutant screening method on fertilized eggs of large yellow croaker

[0095] This embodiment provides an application example of the marine fish gene editing and mutant screening method described in embodiment 1 in large yellow croaker, as follows:

[0096] 1. Synthesis of sgRNA

[0097] (1). sgRNA design

[0098] ①. Determine the target sequence: by referring to relevant literature, find the key gene related to otolith formation or development of large yellow croaker, otol1 gene. Obtain the mRNA sequence of large yellow croaker otol1 gene (XM_010737964.3) from the gene database NCBI. From the CDS region of otol1, select the target point near the downstream of the gene start codon ATG, preferably on the first or second exon. The target sequence is a DNA sequence starting with GG, containing 18 nucleotides in the middle, and ending with nGG (n represents any base), which is the target sequence, and the sequence is 5'-GGT ATATCC TGG TGC GGG ACA GG-3'.

[0099] ②. Design sgRNA: pay attention to avoid high GC content or secondary structure in the target region to improve the cutting efficiency of sgRNA. Ensure that the sgRNA sequence has high specificity, use gene database (such as NCBI BLAST tool), compare the designed sgRNA sequence with the whole genome (or the genome of the target species), and check whether there are other high similarity matching regions except the target region of otol1 gene. If only the target region has high matching degree, it means good specificity, thereby reducing the risk of off-target. The final designed sgRNA sequence is 5'-GGU AUA UCC UGG UGC GGG ACA GGG UUU UAG AGC UAG AAA UAG CAAGUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAAGUG GCACCG AGUCGG UGC UUU UAAA-3'.

[0100] (2). Synthesis of sgRNA

[0101] ①. Strain culture and plasmid extraction:

[0102] Single colonies of *E. coli* DH5α transformed with plasmid pDR274 were picked and inoculated into LB broth containing kanamycin, and cultured overnight at 37°C and 200 rpm. Plasmid pDR274 was extracted using TIANGEN's TIAnypearls Plasmid Extraction MiniKit to obtain a high-purity plasmid with an absorbance ratio (A260 / A280) of 1.80–1.85 and a concentration ≥100 μg / mL.

[0103] ②. Cutting of plasmid pDR274:

[0104] Incubate plasmid pDR274 with BsaI restriction endonuclease at 37°C for 1-2 hours. Take 1 μg of plasmid pDR274, add CutSmart Buffer and BsaI enzyme, and add sterile water to make a total volume of 10 μL. Digest at 37°C for 1 hour to cleave plasmid pDR274 at specific sites, forming a linear plasmid for subsequent ligation reactions.

[0105] ③. Primer annealing:

[0106] The designed sgRNA primers (positive strand 5'-TAG GTA TAT CCT GGT GCG GGAC-3') and negative strand 5'-AAACGT CCC GCACCAGGATAT A-3') were diluted to 10 μM. 5 μL each of the diluted positive and negative primer strands were added to 10 μL of sterile water (PCR water) to make a total volume of 15 μL, with both primer strands at a concentration of 40 μM. The mixture was placed in a PCR instrument and annealed at 95°C for 5 min, then slowly cooled to room temperature to form double-stranded DNA fragments.

[0107] ④. Connection reaction:

[0108] Take 2 μL of the digested pDR274 linear plasmid and add 5 μL of annealed double-stranded DNA fragment (primer-to-ligation vector molar ratio is 3:1). Add 1 μL of T4 DNA ligase and 2× Reaction Buffer, and bring the total volume to 10 μL with sterile water. Incubate the centrifuge tube in a 16°C water bath for 18 hours to allow the double-stranded DNA fragment to ligate with the linear plasmid, forming recombinant pDR274-sgRNA. After incubation, transfer the tube to -20°C for storage until use.

[0109] ⑤. Conversion and Screening:

[0110] The recombinant plasmid pDR274-sgRNA was taken and added to E. coli DH5a competent cells, which were then placed in an ice bath for 30 min, heated at 42°C for 90 s, and then placed in an ice bath for 2 min. LB medium was added, and the mixture was cultured at 37°C and 200 rpm for 1 h. The culture was spread on LB agar plates containing kanamycin, and the plates were incubated at 37°C overnight to screen for positive clones containing the recombinant plasmid pDR274-sgRNA.

[0111] 6. Colony PCR identification:

[0112] A single colony was picked from the plate and inoculated into LB liquid medium containing kanamycin for further culture. The mixture was cultured at 37°C and 200 rpm for 3-5 h. An appropriate amount of bacterial solution was taken, and plasmid extraction was performed according to the instructions of the TIAnypearls Plasmid Extraction Mini Kit. PCR verification was then performed to confirm the correctness of the sgRNA sequence in the recombinant plasmid.

[0113] 7. Plasmid recovery and PCR amplification:

[0114] The TIANprep Rapid Mini Plasmid Extraction Kit was used to extract plasmid from liquid bacterial culture. The kit's instructions were followed, including centrifugation, lysis, neutralization, adsorption, washing, and elution steps, to purify and recover the recombinant pDR274 plasmid. In the PCR reaction system, primers and Q5 Hot Start High-Fidelity DNA Polymerase were added, and the recovered recombinant pDR274 was used as the template for PCR amplification to achieve linearization. The PCR primer sequences were: forward primer 5'-CGC TGTCAAGGA AGA GCACC-3', and reverse primer 5'-AGATGT CCACGC CAC TAC AT-3'. The PCR reaction system contained recombinant plasmid template, primers, dNTPs, Taq polymerase, and buffer components. The amplification conditions were: denaturation at 94°C for 3 min; 25-30 cycles, each cycle including denaturation at 94°C for 30 s, annealing at 55-60°C for 30 s, and extension at 72°C for 1-2 min; and finally extension at 72°C for 5-10 min.

[0115] 7. sgRNA in vitro transcription:

[0116] After confirming the success of linearization by agarose gel electrophoresis, the PCR product was used as a template for in vitro transcription using the MEGAscript T7 Transcription Kit. In the in vitro transcription reaction system, ATP, CTP, GTP, UTP, reaction buffer, linear template DNA and enzyme mixture were added, and the kit instructions were followed. The T7 RNA polymerase catalyzed the transcription of sgRNA at 37°C for 1-2 hours. After the reaction was completed, the transcription product was purified using the MEGAshortscript T7 Transcription Kit, DNase I (RNase-free), DNase I (RNase-free) Stop Solution and 75% ethanol according to the kit instructions, and finally the sgRNA was further purified by ethanol precipitation to obtain sgRNA that can be used for CRISPR-Cas9 gene editing.

[0117] 2. Mixing Cas9 protein and sgRNA

[0118] The Cas9 protein used was a commercial nuclear localization protein. The Cas9 protein was mixed with sgRNA in a buffer containing 10 mM Tris-HCl (pH 7.5), 100 mM KCl, 1 mM DTT to form a ribonucleoprotein (RNP) complex. The ratio of Cas9 protein to sgRNA was 1:3. After gentle mixing, the Cas9 protein and sgRNA were allowed to bind fully at room temperature for 15 minutes. The mixed Cas9-sgRNA RNP complex was diluted to 200 ng / μL before microinjection.

[0119] 3. Microinjection

[0120] (1). Prepare the microinjection needle required for microinjection. The overall length of the needle is 90.00 mm, the outer diameter is 1.00 mm, the inner diameter is 0.60 mm, the needle tip length is 9 mm, and the needle tip opening inner diameter is 0.5 μm. A polished glass needle is used. Prepare the biological glue by adding 4000 CPS methyl cellulose to 1 / 4 of the total water volume of ultrapure water (70°C) and dispersing while using a magnetic stirrer to stir, obtaining a hot gel. Then slowly add the remaining 3 / 4 of the ultrapure water (4°C) and stir on ice to obtain the desired biological glue. Prepare a 2% agarose gel by weighing 0.8 g of agar powder into a conical flask, pouring 40 mL of 1x TAE solution, heating in a microwave oven until melted, pouring into a clean petri dish with 20 mL of agarose solution, and placing at room temperature until solidification. Then place the embryo microinjection mold with the groove facing the already solidified agarose gel, pour the remaining agarose solution from one side of the petri dish onto the mold, and remove the mold after the agarose gel solidifies.

[0121] (2). Use 10 μL of pipette to suck the mixed Cas9-sgRNA RNP complex injection solution and slowly inject it into the injection needle.

[0122] (3). Use a rubber suction tube to select well-shaped fertilized eggs under a body microscope and suck them out with the suction tube. Arrange them in the grooves of the agarose gel in the culture dish one by one, and then suck out the excess water. Then use a suction tube to suck the biological glue that has been prepared and drop it on the fertilized eggs that have been arranged. After completion, stand for five minutes, and then put the culture dish under a body microscope for microinjection.

[0123] (4). Install the injection needle on the mechanical arm of the microinjection operator, fine-tune the culture dish containing the fertilized eggs under the body microscope to the appropriate position, adjust the micro-injection operator, and observe the position of the needle tip to ensure that the needle tip is aligned with the fertilized egg. Then gently pierce the fertilized egg membrane and inject the injection solution under the yolk membrane of the fish near the nucleus. After injection, withdraw the micro-injection needle and proceed to the next injection of the fertilized egg.

[0124] 4. Mutant screening

[0125] (1). Genomic DNA extraction

[0126] ①. Mutant selection:

[0127] When the embryo develops to the point where the otolith of the embryo can be observed under the body microscope to show abnormalities (reduced number of otoliths and smaller volume relative to wild type), the genomic DNA of the mutant embryo can be extracted. As shown in Figure 3 , the otoliths of wild type individuals (WT) are larger in volume, while the otoliths of otol1 mutant individuals show a reduced number and smaller volume.

[0128] ②. Grind the tissue:

[0129] The embryo with abnormal otoliths is picked out with a suction tube and placed in a sterile PCR tube. The fertilized egg is ground into a fine homogenate using a tissue grinder to break the cells and release the genomic DNA.

[0130] ③. Add NaOH solution:

[0131] Add 40 μL of 50 mM NaOH solution to the ground homogenate and mix gently by vortexing. Incubate the mixture in a 95°C water bath for 13 minutes, vortexing gently every few minutes during the incubation. After incubation, immediately cool the mixture on ice.

[0132] ④. Centrifugation and neutralization:

[0133] The cooled mixture was placed in a table centrifuge and centrifuged at 12000-14000 rpm for 3-5 minutes to precipitate the cell debris and impurities to the bottom. The supernatant (about 30-35 μL) was carefully aspirated and transferred to a new sterile centrifuge tube, 4 μL of 1 M Tris-HCl solution (pH 8.0-8.5) was added, and the solution was mixed by gentle vortexing to neutralize the pH and stabilize the DNA. The treated DNA solution was stored in a refrigerator at 4°C until subsequent use.

[0134] (2). PCR and high-resolution melting curve analysis (HRMA) were performed

[0135] PCR system (1 sample): PCR water 2.6 uL, DNA solution 5 uL, forward primer 0.2 uL, reverse primer 0.2 uL, template / sample 2 uL, total volume 10 uL.

[0136] PCR program: denaturation at 95.0°C for 3 minutes; denaturation at 95.0°C for 15 seconds; annealing at 60.0°C for 20 seconds; extension at 72.0°C for 20 seconds; repeat step 2 for 39 times; denaturation at 95.0°C for 10 seconds; melting curve analysis from 65.0°C to 95.0°C at a rate of 0.2°C / s, and plate read was performed.

[0137] The length of the PCR primer should not exceed 150 base pairs, and the optimal length of the product should be less than 100 base pairs, and the specific sequence is as follows: forward primer 5'-CGC TGT CAA GGA AGA GCACC-3', reverse primer 5'-AGATGT CCACGC CACTAC AT-3'.

[0138] Then prepare HRMA: after the PCR reaction is completed, the PCR tube does not need to be opened, and HRMA is directly performed. Ensure that the PCR instrument is equipped with high-resolution melting curve analysis function and has installed the corresponding software for data acquisition and analysis.

[0139] Run HRMA: according to the operation manual of the PCR instrument, start the HRMA program. Perform melting curve analysis at a rate of 0.2°C / s from 65.0°C to 95.0°C, and perform plate read at the same time to monitor the melting of DNA double strands in real time.

[0140] Data analysis: after the completion of HRMA, the melting curve data is analyzed using the matching software. The results are shown in Figure 4

[0141] In terms of melting temperature (Tm), the melting peak of the green curve is less stable than that of the red curve, which is about 86°C. ​

[0142] In terms of the shape of the melting curve, the melting peak of the red curve is relatively narrow, indicating that the melting process is relatively concentrated, which usually means that the secondary structure of the DNA fragment is relatively uniform, or there is less heterogeneity. The melting peak of the green curve is relatively wide, indicating that the melting process is relatively dispersed, which may mean that the secondary structure of the DNA fragment has some heterogeneity, or there are different conformational states.

[0143] In terms of the start and end temperatures of the melting curve, the start temperature of the red curve is around 80℃, and the end temperature is around 90℃. The start temperature of the green curve is around 81℃, and the end temperature is around 91℃.

[0144] In terms of the symmetry of the melting curve, the melting peak of the red curve is relatively symmetrical, indicating that the melting process is relatively uniform. The melting peak of the green curve has some asymmetry, indicating that the melting process may have some irregularities.

[0145] The above results show that the green curve is a mutant individual, and the red curve is a wild-type individual. HRMA analysis can preliminarily distinguish between the two.

[0146] (3). Polyacrylamide gel electrophoresis (PAGE) verification

[0147] After HRMA analysis, some DNA samples (especially those with not very obvious distinction in HRMA analysis) were selected for PAGE verification. PAGE gel was prepared, and the DNA samples were loaded into the wells of the polyacrylamide gel, and 1xTBE buffer was added. Connect the power supply and set the constant voltage to 100V. The electrophoresis time is 1 hour. After electrophoresis, the gel is taken out of the electrophoresis tank, and the DNA is stained with gelred dye, then the electrophoresis result is observed and analyzed under the gel imaging system.

[0148] The results are shown in Figure 5 The length of the DNA changes after the knockout of the mutant gene. After PCR amplification, different length DNA fragments are obtained, resulting in multiple bands of the mutant running out. The wild-type individual has only one band. Figure 5 The last lane of the sample shows a single band, which is a wild-type sample; samples 1-4 show multiple bands, which are mutant samples. PAGE can further verify the HRMA analysis results to ensure accurate screening of mutants.

[0149] This specific embodiment is only an explanation of the present application and is not a limitation of the present application. Any changes made by those skilled in the art after reading the specification of the present application will be protected by the patent law as long as it is within the scope of the claims of the present application.

Claims

1. A method for gene editing and mutant screening of marine fish, characterized in that, The method comprises the following steps: S1. Synthesis of sgRNA: design and synthesize sgRNA for target genes of marine fish; S2. Preparation of Cas9-sgRNA RNP complex: incubate Cas9 protein and sgRNA to form a ribonucleoprotein complex; S3. Microinjection: drop biological glue on the fertilized egg, and then inject the Cas9-sgRNA RNP complex solution into the fertilized egg using a microinjection needle after standing; S4. Screening of mutants: after the fertilized egg develops into an embryo, embryos containing mutant characteristics are picked for genomic DNA extraction, and then the extracted DNA is subjected to PCR and high-resolution melting curve analysis, the mutation is detected by high-resolution melting curve analysis, and the mutant is verified by polyacrylamide gel electrophoresis.

2. The method of sea water fish gene editing and mutant screening according to claim 1, characterized in that, In step S1, the method for designing sgRNA is as follows: a. Determine the target sequence: select a DNA sequence starting with GG, containing 18 nucleotides in the middle, and ending with nGG (n represents any base) on the same exon from the CDS region of the target gene sequence, and then compare it with the marine fish genome to select a sequence with good specificity as the target sequence; b. Design sgRNA: according to the determined target sequence, extract its reverse complementary sequence, and add the sgRNA backbone sequence to the 3' end of the reverse complementary sequence to obtain sgRNA.

3. The method of sea water fish gene editing and mutant screening according to claim 2, characterized in that, In step S1, the method for synthesizing sgRNA is as follows: a. Synthesis of recombinant plasmid: use pDR274 as the template plasmid for synthesizing sgRNA, perform plasmid cutting, sgRNA primer annealing to form DNA double strands, and perform ligation reaction of the cut plasmid and the DNA double strands to construct the recombinant plasmid pDR274-sgRNA; b. In vitro transcription: transform the recombinant plasmid into DH5a, screen positive clones; identify the correctness of the sgRNA sequence in the recombinant plasmid through colony PCR; extract the recombinant plasmid and linearize it; then use the linearized plasmid as a template to transcribe sgRNA and purify it to obtain sgRNA that can be used for CRISPR-Cas9 gene editing.

4. The method of sea water fish gene editing and mutant screening according to claim 3, characterized in that, In step S1, when the marine fish is Pseudosciaena crocea, the target sequence is 5'-GGT ATATCC TGG TGC GGG ACA GG-3'; the sgRNA sequence is 5'-GGU AUA UCC UGG UGC GGG ACA GGG UUU UAG AGC UAG AAA UAG CAA GUU AAA AUA AGG CUAGUC CGU UAU CAACUU GAAAAAGUG GCACCG AGU CGG UGC UUUUAAA-3'; and the sgRNA primer comprises a positive strand with the sequence 5'-TAG GTA TATCCT GGT GCGGGA C-3' and a negative strand with the sequence 5'-AAACGTCCC GCA CCA GGA TAT A-3'.

5. The method of sea water fish gene editing and mutant screening according to claim 1, wherein, In step S2, the ratio of Cas9 protein to sgRNA is 1:(1-5), and the mixture is incubated at room temperature for 15 minutes.

6. The method of sea water fish gene editing and mutant screening according to claim 1, wherein, In step S3, the biological gel is prepared as follows: methyl cellulose with a viscosity of 4000 cps is added to 1 / 4 of the total water volume of 70℃ ultrapure water and dispersed while stirring with a magnetic stirrer to obtain a hot gel, and then the remaining 3 / 4 of 4℃ ultrapure water is slowly added and stirred on ice to obtain the desired biological gel.

7. The method of claim 1, wherein the marine fish gene editing and mutant screening method is characterized by, In step S3, the fertilized egg is placed in the groove of the agarose gel, and then the biological gel is added to the fertilized egg. After completion, it is left to stand for five minutes, and then microinjection is performed.

8. The method of sea water fish gene editing and mutant screening according to claim 1, wherein, In step S3, the microinjection needle is a glass needle with a needle tip length of 9mm and a needle tip opening inner diameter of 0.5μm, which is polished smooth.

9. The method of claim 1, wherein the marine fish gene editing and mutant screening method is characterized by, In step S4, the genomic DNA extraction method is as follows: first, the embryos containing the mutant characterization are ground; Then, NaOH solution is added for alkaline lysis; finally, centrifugation is performed to obtain genomic DNA.

10. The method of sea water fish gene editing and mutant screening as claimed in claim 1, wherein, In step S4, the PCR reaction program is as follows: 95.0℃ denaturation for 3 minutes; 95.0℃ denaturation for 15 seconds; 60.0℃ annealing for 20 seconds; 72.0℃ extension for 20 seconds; cycle to step 2, repeat 39 times; 95.0℃ denaturation for 10 seconds; 65.0℃ to 95.0℃ melting curve analysis, heating rate 0.2℃ / sec, plate read.

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