CN107974466B - Sturgeon CRISPR/Cas9 gene editing method - Google Patents

Sturgeon CRISPR/Cas9 gene editing method Download PDF

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CN107974466B
CN107974466B CN201711282089.5A CN201711282089A CN107974466B CN 107974466 B CN107974466 B CN 107974466B CN 201711282089 A CN201711282089 A CN 201711282089A CN 107974466 B CN107974466 B CN 107974466B
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陈戟
胡红霞
胡炜
朱华
王巍
朱作言
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Beijing Fisheries Research Institute
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Abstract

The invention provides a sturgeon CRISPR/Cas9 gene editing method, which is characterized in that Cas9 protein and target gene gRNA are mixed and injected into a sperm receiving hole of an animal pole of sturgeon 1 cell fertilized eggs with fertilization time not more than 20 minutes by a microinjection method. Mixing 100 ng/muL Cas9 nuclear protein and 30 ng/muL gRNA, introducing the mixture into an animal pole of a sperm-bearing embryo in a cell stage of acipenser ruthenicus 1 by adopting a microinjection method, and obtaining the acipenser ruthenicus embryo with the mutation rate of a target gene as high as 83.1% after hatching, thereby establishing an acipenser ruthenicus target gene accurate editing technology. The acipenser parvus embryos with mutated target genes can be obtained by screening through a PAGE method. The technology can be used for researching the functions of sturgeon genes, can also be used for accurately modifying sturgeon endogenous target genes, and can be used for breeding genetically improved sturgeon breeding new strains.

Description

Sturgeon CRISPR/Cas9 gene editing method
Technical Field
The invention relates to the technical field of aquatic animal genetic breeding and functional gene verification research, in particular to a sturgeon CRISPR/Cas9 gene editing method.
Background
Sturgeon is in the transition period between cartilaginous fish and bony fish in the aspect of biological system evolution, has a multiplicized genome, and has important research value in the aspects of researching fish gene function and evolution. However, studies on gene function in sturgeons have been performed, and the conventional reports are limited to gene cloning and expression pattern analysis (Abdolahnejad et al, 2015.Dong et al, 2015.Fajkowska et al, 2016.Song et al, 2016.Yarmohammadi et al, 2017). Sturgeon fish belongs to large economic fish. Sturgeons are not only delicious in meat quality and comprehensive in nutrition and rich in various essential amino acids and unsaturated fatty acids for human bodies, but also are important caviar production materials (Simeanu et al, 2012). However, wild sturgeon resources have been rapidly declining due to human over-fishing and destruction of aquatic ecosystems, and many sturgeons are even in endangered states (Billard et al, 2000).
Developing the genetic breeding of sturgeons and cultivating sturgeon breeding varieties are key points for meeting the increasing demands of human beings on sturgeon products and protecting wild sturgeon resources. However, sturgeon gene function research and genetic improvement breeding are always limited by the lack of effective technical means.
The gene editing technology is a technology for carrying out site-directed mutation or accurate modification on a genome by utilizing a targeted sequence-specific nuclease, and comprises a Zinc Finger Nuclease (ZFN), a TALE nuclease and a CRISPR/Cas9 system. Gene editing techniques have found wide application in gene function studies and genetic improvement of economic species. Efficient gene editing techniques have been established so far in model fish such as zebrafish, herring and economic fish such as tilapia, rainbow trout, carp, clarias canaliculata, dace semilaevis and finless eel (Doyon et al, 2008; Ansai et al, 2013; Qiu et al, 2014; Li et al, 2014; Yano et al, 2014; Zhong et al, 2016; Qin et al, 2016; Chakrapani et al, 2016; Cui et al, 2017; Feng et al, 2017). However, no gene editing technology has been established in sturgeons to date.
Therefore, the sturgeon gene editing technology is established, and on one hand, the sturgeon gene editing technology can be used for sturgeon functional gene research; on the other hand, the method can be used for exploring and developing a new sturgeon breeding strain, providing a good genetic improvement variety for breeding sturgeons, and protecting sturgeon wild resources.
Disclosure of Invention
The invention provides a sturgeon CRISPR/Cas9 gene editing method, which comprises the steps of constructing a regular Clustered spaced short palindromic repeats (Clustered regular intercalary short palindromic repeats 9, CRISPRs/Cas9) gene editing vector of a target gene, transcribing the gRNA of the target gene in vitro, mixing Cas9 nucleolase protein with the gRNA of the target gene, introducing the mixture into animal polar sperm-receiving pores of fertilized eggs of sturgeon in a 1-cell period, wherein the fertilization time of the animal polar sperm-receiving pores is not more than 20 minutes, and hatching to obtain the sturgeon with the target gene mutation.
In order to achieve the purpose, the invention adopts the following technical measures:
a method of sturgeon CRISPR/Cas9 gene editing comprising: cas9 protein and target gene gRNA are mixed and then injected into a sperm receiving hole of an animal pole of a sturgeon fertilized egg at the 1 cell stage with the fertilization time not exceeding 20 minutes by a microinjection mode;
in the above method, preferably, the sturgeons include, but are not limited to, acipenser baeri (a. bairii), acipenser schrenckii (a. schrenckii), and acipenser russiamensis (a. gueldenstatii);
in the above-described method, preferably, the final concentrations of the Cas9 protein and the target gene gRNA are 100 ng/. mu.l and 30 ng/. mu.l, respectively;
in the above-described method, preferably, the injection volume per fertilized egg is 2 nL.
Compared with the prior art, the invention has the following advantages:
at present, no micromanipulation technology for sturgeon embryos exists, and no technology for accurately editing endogenous genes of sturgeons exists. The invention establishes a sturgeon endogenous gene precise editing technology for the first time, and provides a powerful technical means for developing sturgeon gene function research and sturgeon breeding breed genetic improvement.
The existing system for editing genes, which is a Clustered regularly interspaced short palindromic repeats (CRISPRs/Cas 9), usually adopts a mode of co-injecting Cas9mRNA and guide RNA (gRNA). Since Cas9mRNA is translated into protein and then functions after entering into cells, the intracellular translation efficiency of Cas9mRNA affects the mutation efficiency of endonuclease. The invention adopts a mode of directly injecting the Cas9 protein, omits the process of mRNA translation, and improves the cutting frequency of the Cas9 endonuclease to the target site. Only a gRNA vector of a target gene such as ntl needs to be constructed and transcribed in vitro to synthesize a gRNA. Cas9 protein and gRNA aiming at a gene target site are mixed according to the concentration (final concentration) of 100 ng/mu L and 30 ng/mu L, and are introduced into fertilized eggs of the Acipenser parvum 1 at the cell stage by adopting a microinjection method, the survival rate is 93.9 percent, and the Acipenser parvum embryo with the target gene mutation rate up to 83.1 percent can be obtained, so that the Acipenser parvum target gene precise editing technology is established. The acipenser parvus embryos with mutated target genes can be obtained by screening through a PAGE method. The technology can be used for researching the functions of sturgeon genes, can also be used for accurately modifying sturgeon endogenous target genes, and can be used for breeding genetically improved sturgeon breeding new strains.
Drawings
FIG. 1 shows the partial sequence of the first exon and the gRNA target site of the Acipenser aethiopica ntl gene;
underlined sequences are targets and black triangles indicate the position where Cas9 enzyme cleaves the DNA double strand.
FIG. 2 is an animal pole fertilization hole of fertilized egg of Acipenser parvum 1 at cell stage;
arrows indicate animal polar seminiferous wells.
FIG. 3 is a schematic diagram of the detection of Acipenser parvum ntl gene mutation;
in fig. 3 a: detecting the mutation rate of the target sites of 7 injected embryos; the Hm corresponding band represents the DNA fragment of the target site without mutation, and the Ht corresponding band represents the DNA fragment of the target site with reduced, increased or changed base;
b in FIG. 3: calculating the mutation frequency of the target site according to the Ht band and the Hm band brightness;
c in FIG. 3: the 3 rd and 4 th embryos are tested, and the spine is seen to be bent and the tail is seen to be shortened.
The specific implementation mode is as follows:
the technical solutions of the present invention, if not specifically mentioned, are conventional in the art, and the reagents or materials, if not specifically mentioned, are commercially available. The invention takes the editing of the gene ntl of the acipenser ruthenus as an example, establishes a precise gene editing technology of the endogenous gene of the acipenser ruthenus, and the technology can be used for researching the function of the gene of the acipenser ruthenus and the genetic improvement of the variety of the acipenser ruthenus.
Example 1:
a method for sturgeon CRISPR/Cas9 gene editing, comprising the following steps:
1) obtaining fertilized eggs of Acipenser parvum 1 in a cell stage:
selecting sexual mature acipenser parvum parent fish, and temporarily culturing in an indoor culture system. 36 from artificial reproductionBefore an hour, injecting a mixture of LHR Ha and DOM for artificial induced spawning, wherein the injection dose is 10 mu g LHRHA +1mg DOM/kg body weight, injecting the mixture for 1 time every 12 hours, continuously injecting female sturgeons for 2 times, and injecting male sturgeons for 1 time, extruding semen to a dry beaker when the parent fish has the symptom of semen discharge/ovulation, extruding eggs to a glass plate, and injecting 0.3 × Danieau buffer [17mM NaCl, 2M KCl, 0.12mM MgSO ]4，1.8mM Ca(NO3)2，1.5mM HEPES，pH 7.6]The semen is diluted 100 times for artificial insemination. Fertilization time is 1 minute, and then redundant semen is washed out, so that fertilized eggs of the Acipenser parvum which can be used for microinjection are obtained.
2) Construction of guide RNA for Acipenser parvum target Gene (ntl):
aiming at a target gene ntl (Genebank MG520324) of Acipenser parvifilis, a gRNA target point is designed by using a design website (http:// ZiFiT. paratners. org/ZiFiT /), and is GGCTTGAAGACGTGGATCTT (figure 1). Guide RNA (gRNA) was synthesized according to the methods described in the literature [ Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, journal JK (2013) Efficient genome editing in zebraffing use a CRISPR-Cas system. Nat Biotechnol 31: 227-. The method comprises the following specific steps: two Oligo sequences were synthesized, F1-TAG GCTTGAAGACGTGGATCTT, R1-AAACAAGATCCACGTCTTCAAG. The F1 and R1 primers were mixed at 10mM each, and were first incubated at 95 ℃ for 5 minutes and then slowly cooled to 37 ℃. The DR274 plasmid (Addgene plasmid 42250) was digested with BsaI (NEB, R0535) and ligated to the annealed product. The ligation product was transformed into competent E.coli. PCR was performed using primer M13(-47) and primer F1 to pick out positive clones. The PCR reaction conditions are as follows: 95 ℃ for 2 minutes, 95 ℃ for 15 seconds, 56 ℃ for 15 seconds, 72 ℃ for 30 seconds (30 cycles), 72 ℃ for 5 minutes. Plasmids were extracted by conventional methods. After digestion with DraI (NEB, R0129), a fragment of about 200bp was recovered from the gel, transcribed into gRNA in vitro using MEGAshortscript kit (Ambion, AM1354), and purified. Cas9 protein was purchased from Life Technologies (B25640). Cas9 protein was mixed with gRNAs and a small amount of RNase-free phenol red was added to a final concentration of Cas 9100 ng/. mu.L and gRNAs 30 ng/. mu.L.
3) Fertilized egg of Acipenser parvum in 1 cell stage by microinjection
Placing the fertilized eggs of the Acipenser parvum 1 with the fertilization time not more than 20 minutes prepared in the step 1) in a glass culture dish slightly covered with 0.3 x Da nieau buffer. Acipenser sinensis embryonic animals were adjusted polar up under a stereomicroscope (Olympus, Japan). The mixed solution of Cas9 protein and gRNA was injected into the fertilization well of the fertilized egg animal pole of acipenser baeri 1 cell stage one by one using a nitrogen pressurized quantitative microinjection system (Warner PLI-100A, usa) with an injection volume of 2nL per fertilized egg. The microinjected embryos were cultured at 16 ℃ with a survival rate of 93.9%.
4) Screening of Acipenser parvum embryos with ntl mutated target genes
8 days after fertilization, the microinjected embryos developed to the time of emergence, the embryos were collected individually and genomic DNA was extracted by conventional methods. The gene portion fragment containing the target site was amplified with the detection primers F2-GGAGAGCGAATTTCAGAA and R2-GCGCAATGTCATTT TAATAC. The PCR reaction conditions are as follows: 95 ℃ for 1 min, 95 ℃ for 15 sec, 52 ℃ for 15 sec, 72 ℃ for 30 sec (30 cycles), 72 ℃ for 5 min. Finally, the PCR product was left at 95 ℃ for 1 minute and then slowly cooled to 37 ℃. The final PCR product was electrophoresed on 8% polyacrylamide gel (PAGE) at 80V for 1.5 hours, and the gel was stained with EB. The frequency of mutation at the target site was calculated according to the number of electrophoretic bands and brightness by the method described in the literature [ Chen J, Zhang X, Wang T, Li Z, Guan G, Hong Y (2012) effectiveness detection, qu identification and evaluation of sublle allometric methods. DNASES 19:423-433 ]. According to the detection result, mutation of the target site was detected in 7 microinjected embryos, the highest mutation rate reached 83.1% (fig. 3), and meanwhile, two embryos with higher mutation frequency showed a bent spine and shortened tail phenotype, which is similar to the phenotype of the knocked-down zebrafish ntl gene.
Example 2:
ntl genes of different sturgeons were selected as target genes, target gRNAs were constructed in a conventional manner in the art, and embryo micromanipulation was successfully performed in Siberian sturgeons (A. bairi), Schrenckii, and Russian sturgeons (A. gueldenstii) according to the method in example 1.

Claims (4)

1. A method of sturgeon CRISPR/Cas9 gene editing comprising: cas9 protein and target gene gRNA are mixed and then injected into a sperm receiving hole of an animal pole of a sturgeon fertilized egg at the 1 cell stage with the fertilization time not exceeding 20 minutes by a microinjection mode;
the preparation method of the fertilized egg comprises the following steps: selecting sexual mature sturgeon parent fish, and temporarily culturing in an indoor culture system; injecting a mixture of LHRHA and DOM 36 hours before artificial propagation for artificial spawning induction, wherein the injection dose is 10 mug LHRHA +1mg DOM/kg body weight, injecting the mixture for 1 time every 12 hours, continuously injecting female sturgeons for 2 times, and injecting male sturgeons for 1 time; extruding semen into a dry beaker when the parent fish has the sign of spermiation/ovulation, and extruding eggs into a glass plate; diluting the semen by 100 times by 0.3 times Daniea u buffer for artificial insemination; fertilization time is 1 minute, and then redundant semen is washed away, so that sturgeon fertilized eggs which can be used for microinjection are obtained;
the Danieau buffer is as follows: 17mM NaCl, 2mM KCl, 0.12mM MgSO4，1.8mM Ca(NO3)2，1.5mMHEPES，pH 7.6。
2. A method according to claim 1, wherein the sturgeon comprises acipenser baeri, acipenser baeri (a. baieri), acipenser schrenckii (a. schrenckii), or acipenser russian (a. gueldenstaedii).
3. The method of claim 1, wherein the final concentration of the Cas9 protein and the gRNA of the target gene are 100ng/μ L and 30ng/μ L respectively.
4. The method of claim 1, wherein each zygote is injected in a volume of 2 nL.
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