CN114703232A - A CRISPR/Cas9 gene editing method for red carp - Google Patents

Abstract

A CRISPR/Cas9 gene editing method for red carp, involving biotechnology. Specific steps: identify the target gene in the genome of the red carp by sequence alignment, design the sgRNA target sequence and verification primers, and after the target verification, the target gene sgRNA is synthesized and purified by in vitro transcription; the sgRNA and Cas9 protein are mixed in equal volumes. , Microinjection was carried out on the purse-bag red carp embryos after artificial insemination to knock out the target gene, and DNA was extracted by tail clipping 2 months after the embryos hatched. It provides a new way to realize the rapid trait improvement and genetic selection of the red carp, which is beneficial to further develop the economic value of the red carp and increase the breeding income.

Description

A CRISPR/Cas9 gene editing method for red carp

technical field

The invention relates to biotechnology, in particular to a CRISPR/Cas9 gene editing method for red carp.

Background technique

Carp, as one of the most widely cultured freshwater fish in the world, not only has extremely high edible value, but also its ornamental value is widely recognized by people. The red purse carp, Cyprinus carpio var.wuyuanensis, is a variant of carp, produced in Suiyuan County, Jiangxi Province, China. At the same time, the purse red carp has good hybrid compatibility and can be hybridized with a variety of carp and crucian carp, and the offspring produced have obvious heterosis, which plays an important role in promoting my country's aquaculture industry. Our laboratory has carried out research on the developmental biology and evolutionary mechanism of carp through genomic methods for many years, sequenced, assembled and annotated the genome of the red carp, which provides a basis for the further development of genetic analysis, gene function and genetic breeding of the red carp. Data base.

Compared with traditional fish breeding techniques, such as cross-breeding, CRASPR/Cas9 gene editing technology is more purposeful, time-consuming, and cost-effective to cultivate new varieties, and it can achieve precise editing of target genes and obtain target traits faster. Homozygous mutants, which have certain prospects for improving fish breeding efficiency and improving germplasm resources. At present, CRASPR/Cas9 gene editing technology has been widely used in Nile tilapia, Atlantic salmon and various carps and other aquatic economic species. Breeding and developing new varieties provides valuable reference materials. However, there are no reports of related application research in purse red carp.

The purse red carp is an allotetraploid cyprinid fish with 100 chromosomes. It has undergone the fourth round of whole genome duplication unique to cyprinids. The gene has multiple copies, and the gene sequences of different copies are highly similar. And in the process of evolution, different copies of genes may have new functionalization and subfunctionalization, and this complex relationship brings great challenges to gene editing work. But at the same time, gene editing technology also provides an important means to explore the function and evolutionary fate of different copies of genes in polyploid species. In previous studies, three different copies of the adh8a gene were identified in crucian carp (Dhillon et al, 2018), which is involved in the important metabolic process of acetaldehyde scavenging, and is closely related to the hypoxia tolerance of crucian carp, but it is in the purse red. The copy number and function studies of this gene have not been reported in common carp. Therefore, the establishment of the CRISPR/Cas9 gene editing system in the red carp, and the identification and knockout of the gene can provide a research basis and data for further research and development of new breeding lines of red carp and optimization of the breeding traits of red carp.

SUMMARY OF THE INVENTION

The invention aims to provide a CRISPR/Cas9 gene editing method for red carp, and to provide a new method for its genetic breeding. Using the method of microinjection, the mixed system of the target gene sgRNA and Cas9 protein synthesized in vitro and the phenol red solution was injected into the fertilized egg animal pole of the red carp, and the heterozygous individuals of the red carp with the target gene mutation were obtained by hatching.

In order to achieve the above purpose, the specific steps of the CRISPR/Cas9 gene editing method of the red carp of the present invention are as follows: identifying the red carp adh8a gene through sequence comparison, designing a target gene to knock out the target, and then synthesizing it by in vitro transcription. The corresponding sgRNAs were injected into the animal poles of the fertilized eggs of the purse red carp at the 1-cell stage by microinjection of an appropriate amount of Cas9, sgRNA and phenol red mixed system.

The method for identifying the red carp adh8a gene is as follows: using the ADH8a protein sequence of a reference species to compare the histone sequence of the red carp genome by blastp, and identify the corresponding nucleic acid sequence.

The method for designing a target gene knockout target is: using sgRNACas9 software to design the target, and CasOT software to verify the uniqueness of the target to obtain the target sequence.

The method for synthesizing the corresponding sgRNA is:

Using the target sequence-specific F primer and the universal long primer R with the sgRNA_scaffold sequence, the sgRNA transcription template was first synthesized, and then the sgRNA was transcribed in vitro under the action of T7 RNA transcriptase to synthesize the sgRNA, and finally the sgRNA for knockout was obtained by purification ;

The use of sequence-specific F primers with the target:

5'-GAATTAATACGACTCACTATAGGCCACATGAGTCTCTTCCATGGTTTAAGAGCTATGCTGG-3'

The universal long primer R with sgRNA_scaffold sequence:

5'-AAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTAAACTTGCTATGCTGTTTCCAGCATAGCTCTTAAAC-3';

The method for obtaining the fertilized eggs of the red carp is as follows: under the condition of a water temperature of 24±0.3° C., select sexually mature red carp, and temporarily raise them in an indoor cement pond. The dose of luteinizing hormone-releasing hormone (LHRH-A2) and dione (DOM) was injected for induction of labor, and the injection dose of male fish was half that of female fish; Cloacal hole, gently squeeze the abdomen of the fish to obtain sperm and eggs; mix an appropriate amount of red carp sperm and eggs, sprinkle them into the water, wait for the eggs to be fertilized and sink to the bottom of the water, and collect the fertilized eggs that adhere to the petri dish for display. Microinjection.

In the mixed system of Cas9, sgRNA and phenol red, the final concentration of sgRNA is 1500ng/μL, and the final concentration of Cas9 protein is 1590ng/μL.

The liquid volume of the injected mixed system is 1-2nL.

The invention identifies the target gene in the genome of the red carp through sequence comparison, designs the sgRNA target sequence and the verification primer, and after the target verification, the target gene sgRNA is synthesized and purified by in vitro transcription; after the sgRNA and the Cas9 protein are mixed in equal volumes, Microinjection is carried out on the purse red carp embryos after artificial insemination, and the target gene is knocked out, and DNA is extracted by tail clipping 2 months after the embryo hatches, and the F0 generation mutant heterozygote is obtained through the first-generation sequencing verification. The rapid trait improvement and genetic selection of common carp provide a new approach, which is conducive to further developing the economic value of the red carp and increasing the breeding income.

Description of drawings

Figure 1 shows the gene structure and knockout target of purse red carp adh8a2;

Figure 2 shows the sgRNACas9 software target design interface;

Figure 3 shows the results of sanger sequencing detection of adh8a2 gene knockout in purse red carp.

Detailed ways

The present invention will be further explained below in conjunction with the accompanying drawings and embodiments. The following examples are intended to illustrate the invention, but not to limit the scope of the invention.

The embodiment of the present invention includes the following steps:

1) Mix an appropriate amount of red carp sperm and eggs, and sprinkle them into the water;

2) wait for the egg to sink into the water and adhere to the petri dish;

3) Rinse to remove unfertilized eggs, take out the petri dish, and add an appropriate amount of water, just before the fertilized eggs;

4) Prepare a mixed system of sgRNA and Cas9 protein in a volume of 1:1, incubate at room temperature for 10 min and add a small amount of phenol red as an indicator;

5) Observe the fertilized egg development period under the microscope. After the animal pole appears, microinject the liquid from the previous step. Stop the injection when the embryo develops to the two-cell stage, remove the fertilized eggs in the uninjected area, and add an appropriate amount of methylene blue solution prepared in advance ( 0.1%), culture at 26-28°C, change methylene blue solution once a day, and remove dead eggs in time;

6) When the purse red carp larvae are 2 months old, cut off part of the caudal fin, extract DNA, PCR amplify the genomic target sequence, and carry out Sanger generation sequencing to detect mutants;

The method for obtaining sperm and eggs of red purse carp is as follows: under the condition of water temperature of 24±0.3 ℃, select sexually mature red carp, and temporarily raise them in indoor cement ponds. Dosage injection of luteinizing hormone-releasing hormone (LHRH-A2) and dione (DOM) for induction of labor, the injection dose of male fish is half of that of female fish; when broodstock has ovulation or insemination behavior, dry it with a dry clean towel. Genital hole, gently squeeze the abdomen of the fish to obtain sperm and eggs, and store at 4 °C;

In the mixed system, the final concentration of gRNA is 1500ng/μL, and the final concentration of Cas9 protein is 1590ng/μL;

The injection volume is 1-2nL;

The target gene sgRNA:

GAAUUAAUACGACUCACUAUAGGCCACAUGAGUCUCUUCCAUGGUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUU.

Example 1: Construction of the sgRNA of the red carp target gene (adh8a2)

a) Sequence identification and analysis of the adh8a gene of purse red carp

Download the protein sequences of the adh8a gene of near-source species (zebrafish, medaka, crucian carp) from the official website of NCBI, and organize them into files in fasta format as reference sequences. A library was constructed for the genome sequence of the red carp that was assembled and annotated in our laboratory, and the adh8a1 and adh8a2 genes of the red carp were identified by Tblastn and other analytical methods, and their gene structures were analyzed to obtain the cds sequence of the adh8a2 gene, which was used for gene knockout targets. design. Figure 1 shows the gene structure and knockout target of the red carp adh8a2.

b) Design of target sequence

As shown in Figure 2, the present invention uses sgRNAcas9 software for target design, selects suitable targets according to the result file, and performs target uniqueness verification through CasOT software, and finally obtains the target sequence (adh8a2-target): CCACATGAGTCTCTTCCATG.

c) Design and verification of upstream and downstream primers of target sequence

According to the genomic location information of the red carp where the target sequence is located, a nucleic acid sequence with a length of 1023 bp was extracted from the genome file of the red carp 500 bp upstream of the starting position of the target, and submitted to the NCB online website for target verification primers The upstream primer sequence (adh8a2 target_F) of the obtained target is: CATTTACCCGGTGGGCCTAA, and the downstream primer sequence (adh8a2 target_R) is: GTGTTGACAGCAGCTCCGTA.

d) Synthesis of target sequence sgRNA

The T7 promoter was used to synthesize the target sgRNA, and the sequence of the upstream primer (Primer F) for the synthetic sgRNA was:

5'-GAATTAATACGACTCACTATAGGCCACATGAGTCTCTTCCATGGTTTAAGAGCTATGCTGG-3', the downstream primer is a universal primer (Universal R):

5'-AAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTAAACTTGCTATGCTGTTTCCAGCATAGCTCTTAAAC-3', the upstream and downstream primer sequences were delivered to Jinweizhi Biotechnology Co., Ltd. for synthesis.

The synthesis of sgRNA is divided into three steps:

Step 1: Synthesis of sgRNA In Vitro Transcription Template

Using upstream specific primers and downstream universal primers, TaKaRa high-fidelity enzyme was used for PCR amplification to obtain sgRNA in vitro transcription templates.

reaction system:

Reaction program:

Step 2: In vitro transcription of sgRNA

In a clean bench, the PCR product from the previous step was transcribed in vitro using the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB #E2040).

reaction system:

PCR program: 37℃ 1.5hour

20 μL of enzyme-free water and 2 μL of DNase I (NEB cat#M0303S) were added, and the reaction was carried out at 37°C for 15 min to remove DNA and terminate transcription.

Step 3: Purify sgRNA

The PCR product of the previous step was purified using Monarch RNA Cleanup Kit (NEB T2040L). Add 100 μL RNA Cleanup Binding Buffer and 150 μL pre-cooled absolute ethanol to mix thoroughly with the PCR product of the previous step, then transfer to the adsorption column, centrifuge at 16000×g for 1 min, and discard the liquid; add 500 μL RNACleanup Wash Buffer, Centrifuge at 16000×g for 1 min, discard the liquid; repeat the previous step; in order to reduce the influence of organic reagents, perform another empty tube centrifugation, centrifuge at 16000×g for 1.5 min, and discard the liquid; Transfer the column to a 1.5mL centrifuge tube, add 30μL of nuclease-free water, and centrifuge at 16,000 × g for 1min to obtain sgRNA; take 1uL of sgRNA for agarose gel electrophoresis (2%), 140V, 20min, to detect the integrity of the RNA 1uL sgRNA was used to determine the OD value of the RNA concentration by Nanodrop.

Example 2: Acquisition of fertilized eggs of purse red carp

The male and female broodstock of mature purse red carp were selected and temporarily raised in indoor cement ponds, and the water temperature was controlled at about 24°C. 24h before artificial breeding, the male and female broodstock were injected with a mixed liquid of deonone (DOM) and luteinizing hormone-releasing hormone A2 (LHRH-A2). /kg, the injection dose of male fish is half of the injection dose of female fish, after 12h, the second injection is given to female fish. When the male and female broodstock have spawning/ejaculation, dry the water around the cloaca of the broodstock with a clean towel, squeeze the abdomen gently, and collect the semen and eggs in a clean 50mL centrifuge tube and store at 4°C. Take the appropriate amount of sperm and eggs with a plastic-head dropper into a dry and clean beaker, mix them thoroughly, and sprinkle them evenly into a water basin with a petri dish. After the fertilized eggs settle and adhere to the petri dish, rinse off the unfertilized eggs. Eggs, leaving a small amount of water in a petri dish, no fertilized eggs can be.

Example 3: Microinjection of the target gene of the fertilized egg of the red purse carp

According to the volume ratio of 1:1, mix the sgRNA and Cas9 protein of the target gene, incubate at room temperature for 10 min, add a small amount of phenol red as a color indicator, mix well and pour into a quartz glass needle. Observed under a stereo microscope, the color of the fertilized egg changed from yellow to transparent, and swelled by water absorption, appeared animal pole, and entered the 1-cell stage of embryonic development. In order to ensure the knockout efficiency, microinjection was carried out at the 1-cell stage of the fertilized egg using a nitrogen-pressurized microinjection system, and each fertilized egg was injected with about 2nL of mixed liquid. When the fertilized eggs entered the 2-cell stage, the microinjection was terminated, the uninjected fertilized eggs were removed from the petri dish, and an appropriate amount of 0.1% methylene blue solution was added to prevent the growth of water mold. The injected fertilized eggs were incubated at 24°C, and the dead eggs were removed in time every day, and the methylene blue solution with a concentration of 0.1% was replaced.

Example 4: Screening and detection of target gene mutants of red purse carp

After hatched purse red carp larvae were cultivated to 2 months old, tail clipping was carried out to each fish (18 tails in total), DNA was extracted, and the upstream and downstream primers of c) design in Example 1 were used to carry out PCR amplification target sequence, Perform mutation detection. The specific steps are: take a small amount of caudal fin into a 200 μL centrifuge tube, add 20 μL Extraction Solution (Dongsheng Bio, P9052), briefly centrifuge, put it into a PCR machine, 98°C, 20 min; add 2 μL neutralization Solution (Dongsheng Bio, P9052) , Vortex and mix, and centrifuge briefly to obtain crude DNA; use crude DNA as a template to amplify gene fragments containing target sites.

reaction system:

Reaction program:

Take 2 μL of PCR products for agarose gel electrophoresis (2%), 110V, 35min, and send the remaining PCR products to Primus Biotechnology Co., Ltd. for next-generation sequencing. Sequence analysis of the sequencing results showed that there were two purse red carps with double peaks in the target gene sequence region. The data indicated that the CRISPR/Cas9 gene editing system was successfully established in the purse red carp for the first time, and mutant heterozygotes were obtained.

sequence listing

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Claims (7)

1. A method for editing a Paris polyphylla CRISPR/Cas9 gene is characterized by comprising the following specific steps: firstly, comparing and identifying the Dutch red carp adh8a gene, designing a target gene to knock out a target spot, then synthesizing corresponding sgRNA by an in vitro transcription method, and injecting a proper amount of a mixed system of Cas9, sgRNA and phenol red into an animal polar of Dutch red carp fertilized eggs at a 1-cell stage in a microinjection mode.

2. The method for editing the CRISPR/Cas9 gene of the red purse-red carp as claimed in claim 1, wherein the method for identifying the adh8a gene of the red purse-red carp is as follows: and (3) comparing the protein sequences of the red purse string carp genome with the ADH8a protein sequence of a reference species by blastp, and identifying to obtain a corresponding nucleic acid sequence.

3. The method for editing the CRISPR/Cas9 gene of the red purse-string carp as claimed in claim 1, wherein the method for designing the target gene knockout target is as follows: the sgRNAcas9 software is used for designing a target, and the CasOT software verifies the uniqueness of the target to obtain a target sequence.

4. The method for editing the CRISPR/Cas9 gene of the red purse-string carp as claimed in claim 1, wherein the method for synthesizing the corresponding sgRNA is as follows:

synthesizing a transcription template of the sgRNA by using an F primer with target point sequence specificity and a universal long primer R with a sgRNA _ scaffold sequence, then transcribing the sgRNA in vitro under the action of T7 RNA transcriptase, and finally purifying to obtain the sgRNA for knockout;

the method comprises the following steps of (1) utilizing an F primer with target point sequence specificity:

5‘-GAATTAATACGACTCACTATAGGCCACATGAGTCTCTTCCATGGTTTAAGAGCTATGCTGG-3’

the universal long primer R with sgRNA _ scaffold sequence:

5‘-AAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTAAACTTGCTATGCTGTTTCCAGCATAGCTCTTAAAC-3’。

5. the method for editing the CRISPR/Cas9 gene of the red purse-red carp as claimed in claim 1, wherein the method for obtaining the fertilized egg of the red purse-red carp comprises: under the condition that the water temperature is 24 +/-0.3 ℃, sexually mature red purse carps are selected and temporarily cultured in an indoor cement pond, the parent fishes are injected with luteinizing hormone releasing hormone and diospyrone at the dosage of 3-5 mu g/kg and 1-3 mg/kg for spawning induction, and the injection dosage of the male fishes is half of that of the female fishes; when the parent fish has ovulation or insemination behavior, wiping the cloaca with a dry and clean towel, and slightly squeezing the belly of the fish body to obtain sperms and ova; uniformly mixing a proper amount of sperms and eggs of the red carps under the water, scattering the sperms and the eggs into the water, and collecting the fertilized eggs adhered to a culture dish for microinjection after the eggs are fertilized and sink to the water bottom.

6. The method for editing the CRISPR/Cas9 gene of the red purse-string carp as claimed in claim 1, wherein in the mixed system of Cas9, sgRNA and phenol red, the final concentration of sgRNA is 1500ng/μ L, and the final concentration of Cas9 protein is 1590ng/μ L.

7. The method for editing gene of Netherlands red carp CRISPR/Cas9 as claimed in claim 1, characterized in that the volume of the injected mixed system liquid is 1-2 nL.

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