CN114134150A - 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 SgRNA of a targeted portunus trituberculatus dmy gene and a method for editing the portunus trituberculatus gene. The gene editing method mainly comprises the following steps: (1) SgRNA target design synthesis andCas9preparing mRNA; (2) SgRNA andCas9mixing mRNA; (3) obtaining and microinjecting fertilized eggs of the one-cell stage of the portunus trituberculatus; (4) and (3) detecting the mutation condition of the injected early embryo sample to finally obtain the portunus trituberculatus embryo subjected to gene editing. Meanwhile, the invention also obtains a targeted portunus trituberculatus in the process of target synthesis designdmySgRNA of the gene. The method can realize gene editing of the portunus trituberculatus oosperm, and can lay a foundation for the research of the gene function of the portunus trituberculatus and the cultivation of mutant strains.
Description
Technical Field
The invention belongs to the technical field of gene editing, and particularly relates to SgRNA of a targeted portunus trituberculatus dmy gene and a method for gene editing of portunus trituberculatus.
Background
Portunus trituberculatus (blue crab)Portunus trituberculatus) Belongs to Crustacea, decapod, Paralithodes, and is an important mariculture organism in China. The portunus trituberculatus has rich nutrition and delicious taste, and is deeply popular with consumers at home and abroad. However, according to the national fishery statistics yearbook in 2021, in 2020, the breeding yield of the swimming crabs in the whole country is only 10.1 ten thousand tons, and the yield can not meet the requirements of consumers. The shortage of seedlings is an important reason for restricting the cultivation yield of the portunids. Therefore, the molecular breeding plays an important role in the breeding work of the portunus trituberculatus fries.
CRISPR/Cas9 technology is a technology for gene editing that has emerged in recent years, enabling deletions or insertions to be made to a DNA sequence at the DNA level. However, the technology is not well implemented in the molecular breeding of the blue crabs. Therefore, the development of the CRISPR/Cas9 gene editing technology in the portunus trituberculatus is beneficial to the research of the gene function of the portunus trituberculatus on one hand and the development of a new strain of the portunus trituberculatus on the other hand, and lays a foundation for the cultivation of improved varieties of the portunus trituberculatus.
Disclosure of Invention
The invention provides an SgRNA of a targeted portunus trituberculatus dmy gene and a method for editing the portunus trituberculatus gene, which can realize the gene editing of fertilized eggs of the portunus trituberculatus and further lay a foundation for the cultivation of mutant strains of the portunus trituberculatus.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides a method for editing a portunus trituberculatus gene, which comprises the following steps:
(1) designing and synthesizing a target spot of the SgRNA according to the gene of the portunus trituberculatus, designing and synthesizing a primer according to the target spot of the SgRNA, connecting the gene of the portunus trituberculatus with a plasmid by using the primer, and obtaining the SgRNA after purification and recovery;
(2) after the Cas9 plasmid is subjected to linear enzyme digestion treatment, purification and in vitro transcription are carried out to obtain the recombinant proteinCas9 mRNA；
(3) Mixing the SgRNA in the step (1) and the SgRNA in the step (2)Cas9Diluting mRNA and mixing to obtain injection;
(4) preparing a microinjection mould of the portunus trituberculatus oosperms in advance, and preparing an agarose groove by using the mould;
(5) obtaining portunus trituberculatus germ cells, placing the portunus trituberculatus germ cells in the agarose grooves, injecting the injection in the step (3) into the germ cells, standing, and culturing and maintaining in low-speed seawater to obtain portunus trituberculatus embryos;
(6) extracting genome DNA of the portunus trituberculatus embryo, carrying out PCR amplification cloning, selecting positive clone sequencing to identify the mutation type of the embryo, and obtaining the portunus trituberculatus embryo which is subjected to gene editing.
Further, the nucleotide sequence of the primer in the step (1) is as follows:
F1：TGTAATACGACTCACTATAgagcaaaatgtcaccagtggGTTTTAGAGCTAGAAAT；R1：AAAAAAAGCACCGACTCGGTGCCAC。
further, SgRNA and SgRNA in the step (3)Cas9Diluting mRNA with phenol red and water without enzyme; wherein the dilution concentration of SgRNA is 50 ng/. mu.L,Cas9the diluted concentration of mRNA was 500 ng/. mu.L.
Further, SgRNA and SgRNA in the step (3)Cas9The volume ratio of the mRNA diluted and mixed was 1: 1.
Furthermore, 4-10 ribs with the width of 0.2-0.4 mm and the thickness of 0.2-0.4 mm are arranged in the middle of the microinjection mold; the width of the agarose groove is 0.2 mm-0.4 mm.
Further, the injection amount of the injection in the step (5) is 0.2 nL-0.4 nL per fertilized egg.
Further, in the step (5), injecting the injection into fertilized eggs, standing for 4 hours, placing sterile seawater with the salinity of 25 on a decoloring shaking table at 50rpm for low-speed rotation culture at the temperature of 30 ℃, changing water twice a day, and picking out dead embryos at any time; the seawater was sterilized by filtration using a 0.22 μm filter.
Further, the portunus trituberculatus oosperm is a portunus trituberculatus oosperm 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 in a mode of being watched by multiple persons on duty in turn and observed in real time so as to obtain the fertilized eggs of the portunus trituberculatus which are just discharged, and when the portunus trituberculatus begins to hold eggs, immediately obtaining the fertilized eggs of the portunus trituberculatus in sterile seawater prepared in advance by using a writing brush.
The invention provides a targeted portunus trituberculatus obtained by using the portunus trituberculatus gene editing methoddmyThe nucleotide sequence of the target 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 the microinjection, the groove which is suitable for manufacturing the portunus trituberculatus microinjection agarose and has the width of 0.3mm can be manufactured through the independently designed mould with the specific size and the material, the supporting and protecting functions can be provided for the embryo which is subjected to the microinjection, meanwhile, the embryo can not roll everywhere, and the microinjection efficiency is improved.
2. The invention obtains the mutation sample of the sense mutation type generated by CRISPR/Cas9 gene editing in the portunus trituberculatus fertilized ovum for the first time, and the technical details involved in the invention can provide valuable reference for smoothly developing heritable portunus trituberculatus gene editing operation in the later period and lay a foundation for the cultivation of the improved portunus trituberculatus.
Drawings
FIG. 1 shows Portunus trituberculatusdmyA gene structure schematic diagram and a SgRNA target site; wherein the boxes represent exons and the boxesThe black line between the boxes represents the intron, ATG is the start codon.
Figure 2 is a microinjection mold design.
FIG. 3 is a colony identification PCR electrophoretogram.
FIG. 4 is a drawing showingdmyGene mutation type results.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific examples. The technical scheme of the invention is a routine scheme in the field if not specifically stated, and the used reagents and materials are all from commercial sources if not specifically stated.
The present invention relates to a target genedmy(SEQ ID No.6) as an example, establishes a portunus trituberculatus gene editing technology and lays a foundation for obtaining a viable gene editing portunus trituberculatus embryo 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 SgRNAdmyKnock-out target site of gene: GAGCAAAATGTCACCAGTGGAGG (SEQ ID No.1, FIG. 1).
Primers were designed based on the sequence of the pT7-gRNA plasmid and the target site, and the sequences of the primers were as follows:
F1：TGTAATACGACTCACTATAgagcaaaatgtcaccagtggGTTTTAGAGCTAGAAAT（SEQ ID No.2）；
R1：AAAAAAAGCACCGACTCGGTGCCAC（SEQ ID No.3）。
amplification was performed using the pT7-gRNA plasmid as a template according to the following system:
| 10×Buffer | 5μL |
| 2mM dNTP | 5μL |
| 25mM MgSO4 | 2μL |
| F1 | 1.25μL |
| R1 | 1.25μL |
| pT7-gRNA plasmid | 1μL |
| KOD plus | 1μL |
| ddH2O | 33.5μL |
The amplification conditions were: 3min at 98 ℃; (94 ℃, 30 s; 55 ℃, 30 s; 68 ℃, 20 s). times.30 cycles; at 68 ℃ for 7 min. The resulting PCR product was purified and recovered. The template was transcribed in vitro using the T7 in vitro transcription kit and purified using the RNeasy Mini kit.
2. Preparation of Cas9 mRNA: the Cas9 plasmid is linearized and digested with XbaI, the digestion system is carried out according to the instruction, digestion is carried out for 3h at 37 ℃, and after complete incision is confirmed by electrophoresis, the linearized DNA is 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 of the purified mRNA was measured and the quality thereof was determined by Nanodrop microspectrophotometer and 1.5% agarose electrophoresis, respectively. The SgRNA and Cas9 RNA were diluted with phenol red and enzyme-free water to 50 ng/μ L and 500 ng/μ L, respectively, and then formulated into injected samples at a 1:1 ratio.
Example 2: micro-injection of portunus trituberculatus one-cell stage embryo
Microinjection is carried out on the fertilized eggs of the portunus trituberculatus in the first cell stage.
Preparation in advance is required: a mold with the width of 0.3mm is designed and customized according to the diameter of the fertilized eggs of the portunus trituberculatus (figure 2). The thickness of the die is 0.3mm, the upper part and the lower part of the cross section are straight lines, the left part and the right part are arc edges, and the size of the die is 58mm multiplied by 60 mm; a plurality of ribs with the width of 0.3mm are arranged in the middle of the die; 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. Preparing seawater with salinity of 25, and filtering with 0.22 μm filter membrane for sterilization.
Obtaining fertilized eggs of the portunus trituberculatus in the first cell stage: monitoring the portunus trituberculatus for 24 hours by adopting a mode of multiple persons on duty in turn and observing 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, fertilized eggs of the portunus trituberculatus are obtained in sterile seawater prepared in advance by a writing brush at the first time, and the fertilized eggs of the portunus trituberculatus in the first cell stage are obtained.
The fertilized eggs obtained just before were aspirated with a smooth pipette and gently arranged in an agarose well filled with sterile seawater under a stereomicroscope. The fertilized eggs were injected one by one using a pneumatic pick-up pump, and the sample of example 1 was injected into each fertilized egg at an amount of about 0.2 nL. And (3) after the injected embryo is kept stand for 4 hours, placing the injected embryo on a decoloring shaker with 50rpm by using sterile seawater with the salinity of 25 for low-speed rotation culture, wherein the culture temperature is 30 ℃. The dead embryos were picked up at any time by changing water twice a day.
Example 3: detecting the mutation condition of the injected sample
After 72h of injection, 60 embryos of the injected embryos were taken and genomic DNA was extracted according to the "animal tissue genome extraction kit" of Kangji century Co. Primers were designed to flank the target site with the following sequences:
dmy-F：ACAAAGACTAAACTTAACGGAGAC（SEQ ID No.4）；
dmy-R：GAGAAAGATGGGAAAGGACA（SEQ ID No.5）。
PCR was performed using the 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 |
| ddH2O | 7μL |
The amplification conditions were: 3min at 95 ℃; (95 ℃, 30 s; 55 ℃, 30 s; 72 ℃, 20 s). times.30 cycles; 72 ℃ for 7 min. The obtained PCR product was subjected to 1.5% agarose electrophoresis, and then purified and recovered by using a gel recovery kit (QIAGEN). The DNA fragment was ligated with pmd-18T vector for TA cloning. The ligated PCR product was transformed with DH5a competent. After 12h, the single clones were picked and incubated at 37 ℃ for 2h at 180 rpm. The single clone was identified by PCR, the electrophoretogram is shown in FIG. 3, and the amplified fragment size is 141 bp. 24 positive clones were picked and sequenced with the universal primer M13+ (primer sequence: 5'-GTTTTCCCAGTCACGAC-3', SEQ ID No. 7) (Shanghai, Biotechnology engineering Co., Ltd.). The mutation types were identified by BLAST online software, and the identification results are shown in FIG. 4. As can be seen from the sequencing results, the obtained mutation types were 4, all of which were gene deletion mutations, and the mutation efficiency was 20.8% (mutation efficiency = number of detected mutant clones/total number of successful clones sequenced).
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
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gttttcccag tcacgac 17
Claims (10)
1. A method for editing genes of portunus trituberculatus is characterized by comprising the following steps:
(1) designing and synthesizing a target spot of the SgRNA according to the gene of the portunus trituberculatus, designing and synthesizing a primer according to the target spot of the SgRNA, connecting the gene of the portunus trituberculatus with a plasmid by using the primer, and obtaining the SgRNA after purification and recovery;
(2) after the Cas9 plasmid is subjected to linearization enzyme digestion treatment, the plasmid is purifiedTranscription to obtainCas9 mRNA；
(3) Mixing the SgRNA in the step (1) and the SgRNA in the step (2)Cas9Diluting mRNA and mixing to obtain injection;
(4) preparing a microinjection mould of the portunus trituberculatus oosperms in advance, and preparing an agarose groove by using the mould;
(5) obtaining portunus trituberculatus germ cells, placing the portunus trituberculatus germ cells in the agarose grooves, injecting the injection in the step (3) into the germ cells, standing, and culturing and maintaining in low-speed seawater to obtain portunus trituberculatus embryos;
(6) extracting genome DNA of the portunus trituberculatus embryo, carrying out PCR amplification cloning, selecting positive clone sequencing to identify the mutation type of the embryo, and obtaining the portunus trituberculatus embryo which is subjected to gene editing.
2. The method for gene editing of portunus trituberculatus according to claim 1, wherein the nucleotide sequence of the primers in step (1) is as follows:
F1：TGTAATACGACTCACTATAgagcaaaatgtcaccagtggGTTTTAGAGCTAGAAAT；R1：AAAAAAAGCACCGACTCGGTGCCAC。
3. the method for gene editing of portunus trituberculatus according to claim 1, wherein SgRNA and SgRNA in step (3)Cas9Diluting mRNA with phenol red and water without enzyme; wherein the dilution concentration of SgRNA is 50 ng/. mu.L,Cas9the diluted concentration of mRNA was 500 ng/. mu.L.
4. The method for gene editing of portunus trituberculatus according to claim 1, wherein SgRNA and SgRNA in step (3)Cas9The volume ratio of the mRNA diluted and mixed was 1: 1.
5. The method for gene editing of portunus trituberculatus according to claim 1, wherein the middle of the microinjection mould is provided with 4-10 ribs with the width of 0.2-0.4 mm and the thickness of 0.2-0.4 mm; the width of the agarose groove is 0.2 mm-0.4 mm.
6. The method for gene editing of portunus trituberculatus according to claim 1, wherein the injection amount of the injection in step (5) is 0.2 nL-0.4 nL per fertilized egg.
7. The method for gene editing of portunus trituberculatus according to claim 1, wherein in the step (5), the injection is injected into fertilized eggs, after standing for 4 hours, sterile seawater with the salinity of 25 is placed on a decoloring shaking table at 50rpm for low-speed rotation cultivation, the cultivation temperature is 30 ℃, water is changed twice a day, and dead embryos are picked at any time; the seawater was sterilized by filtration using a 0.22 μm filter.
8. The method for gene editing of portunus trituberculatus according to claim 1, wherein the fertilized eggs of portunus trituberculatus are fertilized eggs 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 in a mode of being watched by multiple persons on duty in turn and observed in real time so as to obtain the fertilized eggs of the portunus trituberculatus which are just discharged, and when the portunus trituberculatus begins to hold eggs, immediately obtaining the fertilized eggs of the portunus trituberculatus in sterile seawater prepared in advance by using a writing brush.
9. Targeted portunus trituberculatus obtained by the method for gene editing of portunus trituberculatus according to any of claims 1-8dmyThe SgRNA of the gene is characterized in that the nucleotide sequence of a target point of the SgRNA is GAGCAAAATGTCACCAGTGGAGG.
10. The targeted portunus trituberculatus of claim 9dmyThe application of the SgRNA of the gene in the gene editing of the portunus trituberculatus.
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