CN114134150B - SgRNA of targeted portunus trituberculatus dmy gene and method for editing portunus trituberculatus gene - Google Patents
SgRNA of targeted portunus trituberculatus dmy gene and method for editing portunus trituberculatus gene Download PDFInfo
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Abstract
The invention discloses a method for editing a portunus trituberculatus gene by targeting SgRNA of the dmy gene of the portunus trituberculatus. The method for editing the genes mainly comprises the following steps: (1) SgRNA target design synthesisCas9mRNA preparation; (2) SgRNACas9mixing mRNA; (3) Obtaining and microinjection of fertilized eggs of portunus trituberculatus in a single cell stage; (4) Detecting mutation condition of the injected early embryo sample to finally obtain the gene edited blue crab embryo. Meanwhile, the invention also obtains a targeted portunus trituberculatus in the target synthesis design processdmySgRNA of the gene. The method can realize gene editing of the fertilized eggs of the portunus trituberculatus and lay a foundation for researching gene functions of the portunus trituberculatus and cultivating mutant strains.
Description
Technical Field
The invention belongs to the technical field of gene editing, and particularly relates to a method for editing a target portunus trituberculatus dmy gene SgRNA and a portunus trituberculatus gene.
Background
Portunus trituberculatus (L.) DCPortunus trituberculatus) Belongs to crustaceans, decapetales, portunidae and portunidae, and is an important mariculture organism in China. The portunus trituberculatus has rich nutrition and delicious taste, and is deeply favored by consumers at home and abroad. However, according to the year's statistical analysis of the fishery in 2021, the national swimming crab culture yield in 2020 is only 10.1 ten thousand tons, which is far from meeting the consumer demand. The shortage of seedlings is an important reason for restricting the cultivation yield of portunids. Therefore, molecular breeding plays an important role in the breeding work of portunus trituberculatus offspring seeds.
CRISPR/Cas9 technology is a technology for gene editing that has emerged in recent years, enabling deletion or insertion of DNA sequences at the DNA level. However, this technique is not well practiced in molecular breeding of portunus trituberculatus. Therefore, the CRISPR/Cas9 gene editing technology is developed in the portunus trituberculatus, which is helpful for researching gene functions of the portunus trituberculatus on one hand and developing new strains of the portunus trituberculatus on the other hand, and lays a foundation for fine breed cultivation of the portunus trituberculatus.
Disclosure of Invention
The invention provides a method for editing a target portunus trituberculatus dmy gene SgRNA and a portunus trituberculatus gene, which can realize gene editing of a portunus trituberculatus fertilized egg so as to lay a foundation for cultivating a portunus trituberculatus mutant strain.
In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:
the invention provides a method for editing portunus trituberculatus genes, which comprises the following steps:
(1) Designing a target point for synthesizing SgRNA according to the gene of the portunus trituberculatus, designing a synthetic primer according to the target point of the SgRNA, connecting the gene of the portunus trituberculatus with a plasmid by using the primer, and purifying and recovering to obtain the SgRNA;
(2) After carrying out linearization enzyme digestion treatment on the Cas9 plasmid, purifying and in vitro transcription to obtainCas9 mRNA；
(3) Combining the SgRNA of step (1) with the SgRNA of step (2)Cas9Diluting mRNA and mixing to obtain injection;
(4) Preparing a microinjection mould of fertilized eggs of portunus trituberculatus in advance, and preparing agarose grooves by using the mould;
(5) Obtaining fertilized eggs of portunus trituberculatus, placing the fertilized eggs in the agarose grooves, injecting the injection in the step (3) into the fertilized eggs, standing, culturing with low-speed seawater, and maintaining to obtain portunus trituberculatus embryos;
(6) Extracting genomic DNA of the blue crab embryo, performing PCR amplification cloning, and selecting positive clone sequencing to identify the mutation type of the embryo, thereby obtaining the blue crab embryo subjected to gene editing.
Further, the nucleotide sequence of the primer in the step (1) is as follows:
F1：TGTAATACGACTCACTATAgagcaaaatgtcaccagtggGTTTTAGAGCTAGAAAT；R1：AAAAAAAGCACCGACTCGGTGCCAC。
further, the SgRNA and the method in the step (3)Cas9The mRNA is diluted by phenol red and enzyme-free water; wherein the dilution concentration of the SgRNA is 50 ng/. Mu.L,Cas9the dilution concentration of mRNA was 500 ng/. Mu.L.
Further, the SgRNA and the method in the step (3)Cas9The volume ratio of the mixture after mRNA dilution was 1:1.
Further, 4-10 ribs with the width of 0.2-mm-0.4 mm and the thickness of 0.2-mm-0.4 mm are arranged in the middle of the microinjection die; the width of the agarose groove is 0.2-mm mm to 0.4mm.
Further, the injection amount of the injection in the step (5) is 0.2 nL-0.4 nL for each fertilized egg.
Further, injecting the injection into fertilized eggs in the step (5), standing for 4 hours, placing sterile seawater with the salinity of 25 on a decoloring shaking table with the speed of 50rpm for low-speed rotation culture, changing water twice a day at the culture temperature of 30 ℃ and picking out dead embryos at any time; the seawater was filter sterilized using a 0.22 μm filter membrane.
Further, the fertilized eggs of the portunus trituberculatus are fertilized eggs of the portunus trituberculatus in a one-cell period; when the fertilized eggs of the portunus trituberculatus in the one-cell period are obtained, monitoring the portunus trituberculatus for 24 hours by adopting a mode of taking a plurality of people on duty and observing in real time so as to obtain the fertilized eggs of the portunus trituberculatus just discharged, and when the fertilized eggs of the portunus trituberculatus are found to start to hold, immediately obtaining the fertilized eggs of the portunus trituberculatus by using a writing brush in sterile seawater prepared in advance.
The invention provides a targeted portunus trituberculatus obtained by using the gene editing method of portunus trituberculatusdmyThe nucleotide sequence of the target spot of the SgRNA of the gene is GAGCAAAATGTCACCAGTGGAGG.
The invention also provides the targeted portunus trituberculatusdmyThe application of the SgRNA of the gene in the gene editing of the portunus trituberculatus.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. in microinjection, the invention can manufacture the groove with the width of 0.3mm which is suitable for the microinjection agarose of the portunus trituberculatus through the mould which is designed independently and has specific size and material, can provide supporting and protecting functions for the embryo which is subjected to microinjection, can also ensure that the embryo can not roll everywhere, and improves the microinjection efficiency.
2. According to the invention, a CRISPR/Cas9 gene editing generated sense mutation type mutation sample is obtained in the fertilized ovum of the portunus trituberculatus for the first time, and the technical details related in the invention can provide valuable references for smoothly carrying out heritable gene editing operation of the portunus trituberculatus in the later period and lay a foundation for fine breed cultivation of the portunus trituberculatus.
Drawings
FIG. 1 is a blue crabdmySchematic gene structure and SgRNA target site; wherein the boxes represent exons, the black lines between boxes represent introns, and ATG is the start codon.
Fig. 2 is a schematic diagram of a microinjection mold.
FIG. 3 is a colony identification PCR electrophoretogram.
FIG. 4 is a diagram ofdmyResults of the type of gene mutation.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to specific embodiments. The technical scheme of the invention is conventional in the field unless specifically stated, and all reagents and materials are commercially available unless specifically stated.
The invention targets genesdmy(SEQ ID No. 6) for example, a portunus trituberculatus gene editing technology is established, and a foundation is laid for obtaining viable gene editing portunus trituberculatus embryos in the later period.
Example 1:dmysynthesis of gene target SgRNA and preparation of Cas9 mRNA
The specific operation steps are as follows:
1. target prediction website (http:// www.crisprscan.org /) design using SgRNAdmyKnockout target site of gene: GAGCAAAATGTCACCAGTGGAGG (SEQ ID No.1, FIG. 1).
Primers were designed based on the pT7-gRNA plasmid sequence and the target site, and the primer sequences were as follows:
F1：TGTAATACGACTCACTATAgagcaaaatgtcaccagtggGTTTTAGAGCTAGAAAT（SEQ ID No.2）；
R1：AAAAAAAGCACCGACTCGGTGCCAC（SEQ ID No.3）。
the pT7-gRNA plasmid was used as a template for amplification according to the following system:
| 10×Buffer | 5μL |
| 2mM dNTP | 5μL |
| 25mM MgSO 4 | 2μL |
| F1 | 1.25μL |
| R1 | 1.25μL |
| pT7-gRNA plasmid | 1μL |
| KOD plus | 1μL |
| ddH 2 O | 33.5μL |
The amplification conditions were: 98 ℃ for 3min; (94 ℃,30s;55 ℃,30s;68 ℃,20 s). Times.30 cycles; and at 68℃for 7min. The PCR product obtained was purified and recovered. The template was transcribed in vitro using a T7 in vitro transcription kit and purified using an RNeasy Mini kit.
2. Preparation of Cas9 mRNA: the Cas9 plasmid was linearized, digested with XbaI, digested with the system in accordance with instructions, digested for 3h at 37℃and the complete cut confirmed by electrophoresis, and the linearized DNA was purified. The linearized plasmid was transcribed in vitro using the SP6 in vitro transcription kit and purified using the RNeasy Mini kit.
3. The concentration measurement and quality identification of the purified mRNA were performed by Nanodrop micro-spectrophotometer and 1.5% agarose electrophoresis, respectively. The SgRNA and Cas9 RNA were diluted to 50 ng/. Mu.L and 500 ng/. Mu.L with phenol red and no enzyme, respectively, and then formulated into injectable samples at a 1:1 ratio.
Example 2: microinjection of blue crab one-cell embryo
Microinjection is carried out on fertilized eggs of portunus trituberculatus in one cell stage.
The preparation is advanced: a mold with a width of 0.3mm (fig. 2) was designed and custom-made according to the diameter of the fertilized eggs of portunus trituberculatus. The thickness of the die is 0.3mm, the cross section is straight up and down, the left and right arc sides are arc sides, and the size of the die is 58mm multiplied by 60mm; a plurality of ribs are arranged in the middle of the die, and the width of the ribs is 0.3mm; the left side and the right side are provided with a 4 multiplied by 6mm high upright post and two ring holes.
The day before injection the grooves were made using a mold and 1.5% agarose and cooled for use. Seawater with a salinity of 25 was prepared and filter sterilized with a 0.22 μm filter membrane.
Obtaining fertilized eggs of portunus trituberculatus in a single cell stage: the portunus trituberculatus is monitored for 24 hours by adopting a mode of alternately watching and observing by a plurality of people in real time so as to obtain fertilized eggs of the portunus trituberculatus which are just discharged. When the portunus trituberculatus begins to hold eggs, the fertilized eggs of the portunus trituberculatus are obtained by a writing brush in the sterile seawater prepared in advance at the first time, so that the fertilized eggs of the portunus trituberculatus in the one-cell period are obtained.
The fertilized eggs just obtained were sucked up with a smooth pipette and gently arranged in agarose grooves with sterile seawater under a stereoscopic microscope. The fertilized eggs were injected one by one using a pneumatic booster pump, and each fertilized egg was injected with the sample of example 1, with a sample size of about 0.2. 0.2 nL. After the embryo after injection is kept stand for 4 hours, the embryo is put on a decoloring shaking table with the salinity of 25 for low-speed rotation culture at the culture temperature of 30 ℃ by using sterile seawater with the salinity of 50 rpm. Changing water twice a day, and picking out dead embryos at any time.
Example 3: detecting mutation of the injected sample
After 72 hours of injection, 60 embryos from the injected embryos were taken and genomic DNA was extracted according to the "animal tissue genome extraction kit" from Kangji corporation. Primers flanking the target site were designed and the sequences were as follows:
dmy-F：ACAAAGACTAAACTTAACGGAGAC（SEQ ID No.4）；
dmy-R：GAGAAAGATGGGAAAGGACA（SEQ ID No.5）。
PCR was performed using genomic DNA as a template. The PCR reaction system is as follows:
| 2×Matermix | 10μL |
| dmy-F | 0.5μL |
| dmy-R | 0.5μL |
| genomic DNA | 2μL |
| ddH 2 O | 7μL |
The amplification conditions were: 3min at 95 ℃; (95 ℃,30s;55 ℃,30s;72 ℃,20 s). Times.30 cycles; the reaction was carried out at 72℃for 7min. The obtained PCR product was subjected to 1.5% agarose electrophoresis, and then purified and recovered using a gel recovery kit (QIAGEN). The DNA fragment was ligated to pmd-18T vector for TA cloning. The PCR products after ligation were transformed with DH5a competence. After 12h, the monoclonal was picked and incubated at 37℃for 2h at 180 rpm. The monoclonal was identified by PCR, and the size of the amplified fragment was 141bp as shown in FIG. 3. The 24 positive clones were picked and sequenced with the universal primer M13+ (primer sequence: 5'-GTTTTCCCAGTCACGAC-3', SEQ ID No. 7) (Shanghai Co., ltd.). The mutation types were identified by BLAST online software, and the identification results are shown in FIG. 4. As a result of sequencing, the obtained mutation types were 4, each of which was a gene deletion mutation, and the mutation efficiency was 20.8% (mutation efficiency=number of detected mutant clones/total number of sequenced successful clones).
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
<110> yellow sea aquatic institute of China aquatic science institute
<120> method for editing gene of Portunus trituberculatus by targeting SgRNA of dmy gene of Portunus trituberculatus
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Claims (7)
1. A method for editing a portunus trituberculatus gene, which is characterized by comprising the following steps:
(1) Designing a target point for synthesizing SgRNA according to the gene of portunus trituberculatus, designing a synthetic primer according to the target point of the SgRNA, and amplifying, purifying and recovering the pT7-gRNA serving as a template by using the primer to obtain the SgRNA;
the nucleotide sequence of the primer is as follows:
F1：TGTAATACGACTCACTATAgagcaaaatgtcaccagtggGTTTTAGAGCTAGAAAT；R1：AAAAAAAGCACCGACTCGGTGCCAC
(2) After carrying out linearization enzyme digestion treatment on the Cas9 plasmid, purifying and transcribing to obtainCas9 mRNA；
(3) Combining the SgRNA of step (1) with the SgRNA of step (2)Cas9Diluting mRNA and mixing to obtain injection;
(4) Preparing a microinjection mould of fertilized eggs of portunus trituberculatus in advance, and preparing agarose grooves by using the mould;
(5) Obtaining fertilized eggs of portunus trituberculatus, placing the fertilized eggs in the agarose grooves, injecting the injection in the step (3) into the fertilized eggs, standing, culturing with low-speed seawater, and maintaining to obtain portunus trituberculatus embryos;
(6) Extracting genomic DNA of the blue crab embryo, performing PCR amplification cloning, and selecting positive clone sequencing to identify the mutation type of the embryo, thereby obtaining the blue crab embryo subjected to gene editing.
2. The method for gene editing of portunus trituberculatus of claim 1 wherein said step (3) comprises the steps of SgRNA andCas9the mRNA is diluted by phenol red and enzyme-free water; wherein the dilution concentration of the SgRNA is 50 ng/. Mu.L,Cas9the dilution concentration of mRNA was 500 ng/. Mu.L.
3. The method for gene editing of portunus trituberculatus of claim 1 wherein said step (3) comprises the steps of SgRNA andCas9the volume ratio of the mixture after mRNA dilution was 1:1.
4. The method for gene editing of portunus trituberculatus as set forth in claim 1, wherein 4-10 ribs with a width of 0.2-mm-0.4 mm and a thickness of 0.2-mm-0.4 mm are provided in the middle of the microinjection mold; the width of the agarose groove is 0.2-mm mm to 0.4mm.
5. The method for gene editing of portunus trituberculatus as in claim 1, wherein the injection amount of the injection in the step (5) is 0.2 nL to 0.4 nL per fertilized egg.
6. The method for gene editing of portunus trituberculatus as in claim 1, wherein in the step (5), the injection is injected into fertilized eggs, after standing for 4 hours, the fertilized eggs are put on a decolorization shaking table with the salinity of 25 for low-speed rotation culture, the culture temperature is 30 ℃ and water is changed twice a day, and dead embryos are picked up at any time; the seawater was filter sterilized using a 0.22 μm filter membrane.
7. The method for gene editing of portunus trituberculatus as set forth in claim 1, wherein the fertilized egg of portunus trituberculatus is a fertilized egg of portunus trituberculatus in one cell stage; when the fertilized eggs of the portunus trituberculatus in the one-cell period are obtained, monitoring the portunus trituberculatus for 24 hours by adopting a mode of taking a plurality of people on duty and observing in real time so as to obtain the fertilized eggs of the portunus trituberculatus just discharged, and when the fertilized eggs of the portunus trituberculatus are found to start to hold, immediately obtaining the fertilized eggs of the portunus trituberculatus by using a writing brush in sterile seawater prepared in advance.
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