Microinjection-based urechis unicinctus gene editing method

Abstract

The invention discloses a microinjection-based urechis unicinctus gene editing method, and belongs to the technical field of gene editing. The method comprises the steps of target spot design, micro-injection, artificial insemination, genotype mutation detection and phenotype mutation detection. According to the knockout method for rapidly obtaining the CRISPR / Cas9 gene with genotype and phenotype mutations, a plurality of targeted sgRNAs are introduced into a CRISPR / Cas9 genome editing system, so that a detection method for generating a target phenotype on F0-generation urechis unicinctus is realized. The urechis unicinctus embryo microinjection operation technology is established for the first time, and gene editing of the urechis unicinctus is successfully achieved. The method provides a powerful technical means for carrying out urechis unicinctus gene function research and genetic improvement of urechis unicinctus breeding varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene editing of aquatic animals, and particularly relates to a method for gene editing of Urechis unicinctus based on microinjection. Background Art

[0002] Urechis unicinctus belongs to the phylum Echiura, class Echiurida, order Xenopneusta, family Urechidae, genus Urechis, commonly known as "sea intestine", and is a common benthic organism in the intertidal zone and subtidal zone along the coasts of the Yellow Sea and Bohai Sea in China. Its meat is delicious, rich in protein, amino acids and trace elements, and has high nutritional and health care value. With the overfishing of wild Urechis unicinctus resources in recent years, the contradiction between market supply and demand has become increasingly prominent. Carrying out genetic breeding of Urechis unicinctus and cultivating new varieties of Urechis unicinctus are the keys to meeting the growing demand of humans for Urechis unicinctus products and protecting wild Urechis unicinctus resources. However, the current research on gene function and genetic improvement of Urechis unicinctus has been restricted by the lack of effective technical means.

[0003] Gene editing technology is the most direct and effective method for studying gene function. In higher animals and model animals, gene editing technology has been widely used in gene function research, from the early TALENs and ZFNs gene editing technologies to the currently widely used CRISPR / Cas9 gene editing technology. Compared with TALENs and ZFNs, the CRISPR / Cas9 gene editing technology has significant advantages such as simple design, rapid construction, high targeting efficiency and operational versatility. The CRISPR / Cas9 gene editing technology has shown broad application potential in many fields such as medicine, agriculture, drug research and development, and animal research, including disease treatment, crop improvement, animal models and biological genetic breeding. Researchers have successfully used the CRISPR / Cas9 technology to achieve gene editing in various animals and plants such as mice, zebrafish, Drosophila, Arabidopsis thaliana, corn, tobacco, nematodes, oysters, and abalones.

[0004] Currently, CRISPR / Cas9 gene editing mainly uses techniques such as microinjection, electroporation, virus-mediated delivery, and gene guns to introduce Cas9 and sgRNA into fertilized eggs. Currently, using microinjection for CRISPR / Cas9 gene editing is the most commonly used method. Although CRISPR / Cas9 gene editing technology has made significant progress in multiple species, its development in the field of marine animals is still relatively slow, limited to a few species and with low editing efficiency. In particular, gene editing has not been achieved in the marine invertebrate Urechis unicinctus. Therefore, the use of microinjection-based CRISPR / Cas9 gene editing technology aims to achieve gene editing in Urechis unicinctus, enabling gene editing technology to be more widely applied to the study of gene functions in Urechis unicinctus and the genetic improvement of cultured varieties. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for gene editing of Urechis unicinctus based on microinjection.

[0006] The present invention adopts the following technical solutions:

[0007] A method for gene editing of Urechis unicinctus based on microinjection, comprising the following steps:

[0008] (1) Target design: Sequencing the genes of Urechis unicinctus to obtain the DNA sequence of the gene regulating the target trait, obtaining the exons and introns of the gene regulating the target trait according to the DNA sequence, designing a suitable knockout target on the exon, and transcribing the sequence of the target in vitro into sgRNA;

[0009] (2) Microinjection: Spread the eggs of Urechis unicinctus on a gel groove made of agarose, add an appropriate amount of FSW (sterile seawater) to prevent drying during injection, mix cas9 protein and sgRNA at a molar ratio of 1.7:1, incubate at 37°C for 5 min to form an RNP complex, and then perform microinjection on the eggs of Urechis unicinctus;

[0010] (3) Artificial insemination: Take an appropriate amount of sperm from male Urechis unicinctus and add it to the injected eggs and mix well, ensuring that the process from egg injection to fertilization does not exceed 2 hours. The obtained Urechis unicinctus is the F0 generation chimeric Urechis unicinctus with the trait after knocking out the target gene;

[0011] (4) Genotype mutation detection: Extract the genomic DNA of a single larva after gene editing, PCR amplify the target fragment, observe the size of the target band by gel electrophoresis, and perform sanger sequencing to obtain genotype mutations with long fragment insertions or deletions;

[0012] (5) Phenotypic mutation detection: The expression of the target gene in the gene-edited larvae was detected by in situ hybridization, and the gene-edited larvae were observed under an optical microscope. The mosaic mutation phenotype could be observed in the F0 generation.

[0013] Preferably, in step (1), after obtaining the target sequence, before in vitro transcription, a local database of the Echinops monocyclica genome is first constructed, and blast software is used to search and determine the specificity of the target to avoid off-target knockout.

[0014] Preferably, in step (2), the final concentration of the target gene sgRNA is 300 ng / μL, the final concentration of the Cas9 mRNA is 510 ng / μL, and the injection volume of each egg is 1 nL.

[0015] Preferably, in step (3), the injected fertilized eggs need to be incubated in filtered seawater for 24 hours, and after the monocyclic worms develop to the early trochophore larvae stage and show obvious target traits, frozen samples are taken from the surviving individuals.

[0016] Preferably, in step (4), the DNA extraction and Sanger sequencing of a single larva are specifically as follows: the selected fertilized eggs are placed in a PCR tube, 20 μL of freshly prepared genomic DNA extraction buffer is added, and the reaction is carried out at 55° C. for 3 hours in a PCR instrument and used as a DNA template; PCR amplification primers are designed near the knockout target site, and after PCR amplification, transformation, and cloning, 10 target clones are selected for sequencing and compared with normal sequences to verify whether the knockout site is successfully knocked out.

[0017] Preferably, in the step (5): by comparing the results of the whole-body in situ hybridization experiment between the mutant and the wild type, the signal at the perioral ciliary ring and the apical cilia of the wild type is intact, while the signal at the perioral ciliary ring and the apical cilia of the mutant larvae is weakened or even disappears. This result strongly proves the effectiveness of the gene editing method of the present invention based on microinjection of the monocyclic worm.

[0018] Preferably, in step (5), by comparing the results of optical microscope observation of the mutant and the wild type, the cilia of the mutant individuals become shorter or even disappear, and the swimming speed of the mutant individuals is significantly reduced compared with the wild type.

[0019] Advantages of the technical solution of the present invention:

[0020] At present, there is no report on the application of CRISPR / Cas9 gene editing technology to A. monocygnus. The present invention successfully introduced sgRNA and Cas9 protein into A. monocygnus eggs by microinjection, and finally obtained A. monocygnus gene mutation.

[0021] The CRISPR / Cas9 gene editing technology for Urechis unicinctus was established for the first time, providing a powerful technical means for further research on the gene function and genetic improvement of Urechis unicinctus in the future. The present invention can achieve precise editing of the target gene of Urechis unicinctus. Moreover, the method of re-fertilization after injecting eggs is used in the present invention to enable the RNA to be translated into protein earlier to produce an effect, thereby improving the gene editing efficiency. The injection efficiency is greatly improved by adopting the method of arranging and injecting single eggs; the best final concentrations of sgRNA and Cas9 are used in microinjection, which can improve the survival rate and editing efficiency of the injected Urechis unicinctus larvae, laying a foundation for the research on the gene function and genetic improvement of Urechis unicinctus by using the CRISPR / Cas9 gene editing method. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Agarose gel plate used for injection (left) and injection process (right);

[0023] Figure 2 Deletion situation of the target gene sequence after gene editing;

[0024] Figure 3 Change of peak map after gene editing;

[0025] Figure 4 Phenotype map of whole mount in situ hybridization of caveolin-1;

[0026] Figure 5 Phenotype maps of wild-type and mutant larvae;

[0027] Figure 6 Movement trajectory maps (A) of wild-type and mutant larvae and difference map (B) of swimming speed of larvae; DETAILED DESCRIPTION OF THE INVENTION

[0028] For the convenience of understanding, the technical solutions of the present invention will be described more specifically below with reference to the embodiments.

[0029] Embodiment

[0030] Taking the caveolin-1 gene of Urechis unicinctus as an example, the gene editing technology for Urechis unicinctus was established, and the specific steps are as follows:

[0031] (1) Construction of sgRNA of caveolin-1 gene of Urechis unicinctus

[0032] ① Design of sgRNA primers

[0033] The nucleic acid sequence of the caveolin-1 gene of Urechis unicinctus is shown in SEQ ID NO:1:

[0034] SEQ ID NO:1

[0035]

[0036] According to the sgRNA design principle, 4 sgRNA primers were designed for 4 targets on the second exon of the Urechis unicinctus caveolin-1 gene, and the nucleic acid sequences are as follows:

[0037] sgRNA1 primer: 5’-CCTCTAATACGACTCACTATAGGGACCTACGAGGAGGTAC

[0038] TGGGTTTAAGAGCTATGC-3’ (SEQ ID NO:2);

[0039] sgRNA2 primer: 5’-CCTCTAATACGACTCACTATAGGGAGCCATTGGGATACCA

[0040] CACAGTTTAAGAGCTATGC-3’ (SEQ ID NO:3);

[0041] sgRNA3 primer: 5’-CCTCTAATACGACTCACTATAGGGCAACCCCAGCAGATAG

[0042] CCATGTTTAAGAGCTATGC-3’ (SEQ ID NO:4);

[0043] sgRNA4 primer: 5’-CCTCTAATACGACTCACTATAGGGCAAGGTGTGAGACAA

[0044] GCATGTTTAAGAGCTATGC-3’ (SEQ ID NO:5);

[0045] ② Amplification and purification of sgRNAs DNA template

[0046] Use the sgRNA in vitro transcription kit (Takara, 632636) to synthesize the sgRNAs DNA template.

[0047] Reaction system: 11 μL of RNase Free Water, 12.5 μL of PrimeSTAR Max Premix (2X), 1 μL of Guide-it Scaffold Template, 0.5 μL of Fprimer (10 μM);

[0048] The PCR reaction conditions are: 33 cycles including 98°C for 10 s and 68°C for 10 s; 4°C forever.

[0049] ③ In vitro transcription of sgRNA

[0050] Using the PCR product in ② above as a template, transcribe sgRNA using an in vitro transcription kit for sgRNA (Takara, 632636), and purify sgRNA using an RNA purification kit (Takara, 632638). The average concentration of the purified sgRNAs is approximately 2000 ng / μL.

[0051] (2) Fabricate a fixation mold

[0052] When fabricating a solid seawater agar mold, pour the heated liquid 0.8% seawater agar into a disposable plastic Petri dish with a diameter of 50 mm. When the agar has not completely solidified, place the capillary needle in the Petri dish and press it with a hard paper sheet to form an arc-shaped groove.

[0053] (3) Microinjection

[0054] Mix the target gene sgRNA and Cas9 protein, and incubate at 37 °C for 5 min. Mix the mixture with an equal volume of 0.07% phenol red solution; the final concentration of each of the 4 sgRNAs is 300 ng / μL, and the final concentration of Cas9 protein is 510 ng / μL.

[0055] Arrange the Urechis unicinctus eggs one by one in the arc-shaped groove prepared in step (2), add an appropriate amount of sterile seawater to prevent drying during injection, and the injection volume for each egg is approximately 1 nL ( Figure 1 ).

[0056] (4) Collection and fertilization of eggs

[0057] Place the injected eggs in sterile seawater at 17 - 19 °C for culture, drop in an appropriate amount of sperm, and after incubating for 24 h, collect the Urechis unicinctus larvae.

[0058] (5) Genotype mutation detection

[0059] Adopt the method of extracting genomic DNA from a single larva to extract the genomic DNA of the larvae to be detected.

[0060] Design genotype detection primer sequences on both sides of the 4 sgRNAs as follows:

[0061] test F: 5’-ATGGGTTTGAAAGAAGCTTGGGA-3’ (SEQ ID NO:6)

[0062] test R: 5’-CTATTTCTTGAAGGCGTTGAAGCA-3’ (SEQ ID NO:7).

[0063] Using the larval DNA to be genotyped as a template and test F and test R as primers, amplify the gene fragment containing the target site. After ligation, transformation, and cloning, pick monoclonal colonies and send them to a sequencing company for sequencing.

[0064] Detect long - fragment deletion mutations, long - fragment insertion mutations, and single - base mutations ( Figure 2 ), and compare the peak maps of wild - type and mutant types ( Figure 3 ).

[0065] (6) Phenotypic mutation detection

[0066] Collect the edited larvae and perform in - situ hybridization experiments on the caveolin - 1 gene. The results are shown in Figure 4 . For wild - type larvae, the signals at the peristomial ciliary ring and apical cilia are complete. For mutant larvae, the signals at the peristomial ciliary ring and apical cilia are weakened or even disappear. Detect the mosaic mutation phenotype of mutants with the aid of an optical microscope. The results are shown in Figure 5 and 6 . For wild - type larvae, the cilia are complete. For mutant larvae, the cilia become shorter or even disappear, and the swimming speed of mutant larvae is significantly lower compared with that of wild - type larvae.

[0067] The above results show that using the single - ringed sea cucumber CRISPR / Cas9 gene - editing method provided by the present invention to mediate the target gene to generate long - fragment deletion mutations, the present invention can achieve precise editing of the target gene of the single - ringed sea cucumber. Moreover, the present invention uses the method of injecting eggs and then fertilizing them, allowing RNA to be translated into proteins earlier to produce effects, thereby improving the gene - editing efficiency. The method of arranging and injecting single eggs greatly improves the injection efficiency; using the optimal final concentrations of sgRNA and Cas9 in microinjection can improve the survival rate and editing efficiency of single - ringed sea cucumber larvae after injection, laying a foundation for the study of gene function and genetic improvement of single - ringed sea cucumber using the CRISPR / Cas9 gene - editing method.

[0068] The above - mentioned embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for editing the gene of Echinops monocytogenes based on microinjection, characterized in that: The following steps are involved: (1) Target design: Gene sequencing is performed on the monocyclic spiny worm to obtain the DNA sequence of the gene regulating the target trait, and the exons and introns of the gene regulating the target trait are obtained based on the DNA sequence, and a suitable knockout target is designed on the exon, and the sequence of the target is transcribed in vitro into sgRNA; (2) Microinjection: Spread the eggs of the monocyclic worm flat in a gel groove made of agarose, add an appropriate amount of FSW (sterile seawater) to prevent drying during the injection process, mix the cas9 protein and sgRNA at a molar ratio of 1.7:1, incubate at 37°C for 5 minutes to form an RNP complex, and then microinject the eggs of the monocyclic worm; (3) Artificial insemination: Take an appropriate amount of sperm from male Echinops unicyclicus and add it to the injected eggs and mix well, ensuring that the process from egg injection to fertilization does not exceed 2 hours. The resulting Echinops unicyclicus is the F0 generation chimeric Echinops unicyclicus with the trait that appears after the target gene is knocked out; (4) Genotype mutation detection: Extract the genomic DNA of a single larva after gene editing, amplify the target fragment by PCR, observe the size of the target band by gel electrophoresis, and perform Sanger sequencing to obtain the genotype mutation of long fragment insertion or deletion; (5) Phenotypic mutation detection: The expression of the target gene in the gene-edited larvae was detected by in situ hybridization, and the gene-edited larvae were observed using an optical microscope. The mosaic mutation phenotype could be observed in the F0 generation.

2. The method for editing the gene of Echinops monocyclica based on microinjection according to claim 1, characterized in that: In the step (1), after obtaining the target sequence, before in vitro transcription, a local database of the Echinops monocyclica genome is first constructed, and blast software is used to search and determine the specificity of the target to avoid knockout of off-target.

3. The method for editing the gene of Echinops monocyclica based on microinjection according to claim 1, characterized in that: In the step (2), the final concentration of the target gene sgRNA is 300 ng / μL, the final concentration of the Cas9 mRNA is 510 ng / μL, and the injection volume of each fertilized egg is 1 nL.

4. The method for editing the gene of Echinops monocyclica based on microinjection according to claim 1, characterized in that: In the step (3), the injected fertilized eggs need to be incubated in filtered seawater for 24 hours, and after the monocyclic worms develop to the early trochophore larvae stage and show obvious target traits, cryopreservation samples are taken from the surviving individuals.

5. The method for editing the gene of Echinops monocyclica based on microinjection according to claim 1, characterized in that: In the step (4), the DNA extraction and Sanger sequencing of a single larva are specifically as follows: the selected fertilized eggs are placed in a PCR tube, 20 μL of freshly prepared genomic DNA extraction buffer is added, and the reaction is carried out at 55° C. for 3 hours in a PCR instrument and then used as a DNA template; PCR amplification primers are designed near the knockout target site, and after PCR amplification, transformation, and cloning, 10 target clones are selected for sequencing and compared with the normal sequence to verify whether the knockout site is successfully knocked out.

6. The method for editing the gene of Echinops monocyclica based on microinjection according to claim 1, characterized in that: In the step (5): by comparing the results of the whole-body in situ hybridization experiment between the wild type and the mutant, the signal at the perioral ciliary ring and the apical cilia of the wild type is intact, while the signal at the perioral ciliary ring and the apical cilia of the mutant larvae is weakened or even disappears. This result strongly proves the effectiveness of the gene editing method of the present invention based on microinjection of the monocyclic worm.

7. The method for editing the gene of Echinops monocyclica based on microinjection according to claim 1, characterized in that: In the step (5), by comparing the results of optical microscope observation of the mutant and the wild type, the cilia of the mutant individuals become shorter or even disappear, and the swimming speed of the mutant individuals is significantly reduced compared with the wild type.

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