CN114934076A - Preparation method and application of zebra fish hoxba gene cluster deletion mutant - Google Patents

Preparation method and application of zebra fish hoxba gene cluster deletion mutant Download PDF

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CN114934076A
CN114934076A CN202210760774.9A CN202210760774A CN114934076A CN 114934076 A CN114934076 A CN 114934076A CN 202210760774 A CN202210760774 A CN 202210760774A CN 114934076 A CN114934076 A CN 114934076A
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hoxba
gene cluster
grna
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祖尧
何宏阳
胡沛男
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Shanghai Ocean University
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Abstract

The invention discloses a preparation method and application of a zebra fish hoxba gene cluster deletion mutant, which comprises the following steps: determining that knockout targets of the hoxba gene cluster are respectively located on the 1 st exon of the hoxb13a gene and the 1 st exon of the hoxb1a gene to design gRNA sequences, performing PCR amplification by using a gRNA backbone plasmid as a template, performing PCR product purification and in vitro transcription to obtain gRNAs, mixing the gRNAs of the hoxb13a and the hoxb1a with a Cas9 protein, introducing the mixture into zebra fish, and culturing to obtain the stably inherited hoxba gene cluster deletion mutant. The invention provides a method for knocking out a plurality of continuous genes on a genome together and obtaining a stable mutant, and meanwhile, the phenotype of the mutant lays a foundation for researching the biological function of the hoxba gene cluster and diseases related to the deletion of the hoxba gene cluster.

Description

Preparation method and application of zebra fish hoxba gene cluster deletion mutant
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a preparation method and application of a zebra fish hoxba gene cluster deletion mutant.
Background
The CRISPR-Cas9 is a third-generation gene editing technology after the gene editing technologies such as ZFN, TALENs and the like are introduced, and in a few years, the CRISPR-Cas9 technology is popular in the world, becomes one of the existing technologies with high efficiency, simplicity, low cost and easy operation in gene editing and gene modification, and becomes a mainstream gene editing system at present. The CRISPR/Cas9 system is based on a prokaryotic immune mechanism, can prevent nucleic acid invasion, and belongs to an adaptive immune defense mechanism of bacteria and archaea. It is produced during the course of the continuous evolution of organisms and is used to protect their own genome from interference by foreign nucleic acids. As early as 1987, discovered by researchers at the University of Osaka (Osaka University), in 2012, Doudna and Charpentier combined with each other to modify the CRISPR/Cas system of Streptococcus pyogenes of WT to integrate tracrRNA and crRNA into a single strand, which is called crRNA: tracrRNA chimera, i.e., the present gRNA. The activity of the Cas9 can be started by complementary pairing of the chimeric RNA and the corresponding sequence of the target DNA, and the DNA is cut, so that the research lays a foundation that the CRISPR/Cas becomes a gene editing tool, and the CRISPR/Cas also becomes a gene editing technology with more applications in model animal zebra fish due to the convenience of operation. In 2013, scientists worked to implement the CRISPR/Cas system for genome editing in human cells. Cas 9-mediated gene editing step: firstly, the Cas9 endonuclease cuts genomic DNA under the mediation of gRNA, and Double Strand Break (DSB) occurs in DNA; the fragmented DNA will then be repaired by the cell's own DNA repair system.
Zebrafish have made progress in developmental biology, oncology, toxicology, reproductive research, genetics, neurobiology, environmental science, stem cell research, regenerative medicine, and other fields because of their small size and rapid growth. Zebrafish, as the best model animal in developmental genetics, are powerful tools to assist in the study of the human genome. Zebra fish is also a good model for drug discovery and development, has been widely applied to human disease model research, new drug screening, drug toxicity and safety evaluation, and currently has more than 8000 mutants of zebra fish, wherein about 1/4 becomes a human disease model, and is an ideal gene editing tool at present. At present, multiple genes or small fragment gene knockout is carried out by using multiple gRNAs and Cas9 together in organisms such as yeast, rice, mice and the like, but few reports are carried out on the deletion of fragments of more than 100kb in zebra fish by using a CRISPR/Cas9 technology.
The Hox gene is a large family of transcription factors containing homeoboxes, the full name of homeotic genes. They are arranged in clusters on chromosomes and are distributed on different chromosomes. The Hox gene family controls the construction of body morphology during early embryonic development, and mutation of the Hox gene often causes corresponding deformity of corresponding parts of the body. Related reports show that the Hox gene plays an important role in the development and formation of vertebrate hearts. Medina-Martinez et al firstly constructed a mouse mutant strain with a HoxB gene cluster large fragment deletion of 90kb in total (except Hoxb 13) of Hoxb1-Hoxb9 by using a homologous recombination gene knockout method in 2000, but the whole HoxB gene cluster is not completely knocked out by the mouse strain, and a specific mechanism of an abnormal heart phenotype of the mouse strain is to be deeply analyzed. It has also been found that clinical patients lack part of the HoxB gene cluster (HoxB1-HoxB9) or multiple HoxB genes, and thus have a series of clinical manifestations of growth retardation, cardiopulmonary-cerebral dysplasia, abnormal hands and feet, congenital facial deformity, etc., with the most serious of the patients dying in the four months after birth. However, these studies do not deeply explore the downstream regulatory pathways of the gene, and further elucidation and research are needed.
Disclosure of Invention
The invention mainly aims to provide a preparation method of zebra fish hoxba gene cluster deletion mutants, which utilizes zebra fish as a model organism, designs a target sequence through a CRISPR/Cas9 technology, carries out gene editing on the hoxba gene cluster to realize the targeted knockout of a target gene, and provides a construction thought for commonly knocking out a plurality of continuous genes on a genome to obtain stable mutants.
The invention also aims to provide application of the zebra fish hoxba gene cluster deletion mutant as an animal model in researching the biological function of the hoxba gene cluster and diseases related to hoxba gene cluster deletion, wherein the hoxba cluster plays an important role in heart development, obvious defects can be shown on the heart when the gene is deleted, and the animal model constructed by the zebra fish hoxba gene cluster deletion mutant can provide a mode and a method for researching related diseases such as abnormal heart development and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of a zebra fish hoxba gene cluster deletion mutant, which comprises the following steps:
s1, determining a hoxba gene cluster knockout target to design gRNA sequences on the 1 st exon of zebrafish hoxb13a gene and the 1 st exon of hoxb1a gene;
s2, designing an upstream primer T7-hoxb13a-sfd, T7-hoxb1a-sfd and a downstream gRNA reverse primer for synthesizing gRNA;
s3, respectively carrying out PCR amplification by using primers T7-hoxb13a-sfd, T7-hoxb1a-sfd and a downstream gRNA reverse primer by using a gRNA framework plasmid as a template;
s4, carrying out in vitro transcription on the PCR product obtained in the step S3, and transforming to obtain hoxb13a gRNA and hoxb1a gRNA;
s5, mixing the hoxb13a gRNA and the hoxb1a gRNA with the Cas9 protein, and then performing microinjection and introduction into a zebra fish one-cell-stage embryo;
s6, culturing to obtain the stably inherited zebra fish hoxba gene cluster deletion mutant.
Preferably, in step S1, the target gRNA sequence is
hoxb13a：GGATGAGCTGAAGAATATGG(SEQ ID NO:1)
hoxb1a：GGAACTGGGACAACAAGTTA(SEQ ID NO:2)
Preferably, in step S2, the upstream primer F1
Figure BDA0003724216080000031
Namely the sequence of the primer T7-hoxb13a-sfd is
Figure BDA0003724216080000032
The sequence of primer T7-hoxb1a-sfd is
Figure BDA0003724216080000033
The sequence of a downstream primer R1(trans reverse), i.e., a gRNA reverse primer, is
AAAAAAAGCACCGACTCGGTGCCAC(SEQ ID NO:5)。
Preferably, in step S4, the sequence of hoxb13a gRNA is
TAATACGACTCACTATAGGATGAGCTGAAGAATATGGGTTTTAGAGCTAGAAATAGCGGACAGATTCATGTCCTGGACGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO:6)。
The sequence of the hoxb1a gRNA is
TAATACGACTCACTATAGGAACTGGGACAACAAGTTAGTTTTAGAGCTAGAAATAGCGGACAGATTCATGTCCTGGACGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO:7)。
Preferably, in step S5, the final concentrations of both hoxb13a gRNA and hoxb1a gRNA are 100ng/μ L, the final concentration of Cas9 protein is 800ng/μ L, and the total volume is 1 nL.
Preferably, step S6 specifically includes the following steps:
a1, respectively taking zebra fish with gRNA and Cas9 protein introduced and wild-type zebra fish embryos which are not injected for hoxba gene cluster knockout detection, and determining hoxba gene cluster knockout positive F 0 Culturing to adult fish;
a2, knocking out hoxba gene cluster to be positive F 0 Carrying out heritability and effective mutation detection on the adult fish and wild zebra fish outcrossing, and screening heritability effective mutation F 1 Feeding to adult fish, and obtaining hoxba gene cluster F through genotype identification 1 Mutant zebrafish;
a3, cluster F of the same mutated hoxba gene 1 Carrying out internal crossing on mutant zebra fish to obtain a hoxba gene cluster F 2 Mutant zebrafish;
a4, identified as F 2 Homozygotes of the deletion of the hoxba gene cluster in the generations, namely stably inherited zebrafish hoxba gene cluster deletion mutants.
Preferably, in step a1, the detection primers used for the hoxba gene cluster knockout detection include:
the upstream primer hoxb13 a-F: CGTCACGTGGTACTGCTCTC (SEQ ID NO: 8);
the downstream primer hoxb1 a-R: CAATCCACCTGTTTTGGGGG (SEQ ID NO: 9);
the upstream primer hoxb1 a-F: ACGCTGATGGACGACTTTACG (SEQ ID NO:10)
More preferably, step S6 includes the steps of:
(1) designing primers around the target point, so that the distance between the primers and the two sides of the target point is greater than 100bp, and the absolute value of the difference of the distances between the primers and the target point is greater than 100 bp; selecting a pair of healthy WT zebra fishes as parents, cutting tails to perform PCR, directly sequencing PCR products, detecting whether genes to be knocked out of parent fishes are homozygotes, requiring that target sequences of adult fishes to be injected are homozygotes, and if sequencing results show that the target sequences are heterozygotes, reselecting the adult fishes to be injected;
(2) microinjecting gRNA and Cas9 protein into zebra fish, and mixing the gRNA with the final concentration of 100 ng/mu L of an injection system; cas9 protein: 800 ng/mu L, and the total volume is 1 nL;
(3) picking out dead eggs in the evening on the injection day, changing half of fresh water, changing water once in the morning and at the evening, 48h after fertilization, detecting double-outer-side primer PCR, and knocking out successfully F 0 Feeding zebra fish;
(4) after 3-4 months of sexual maturity of zebra fish, mutating F 0 Hybridizing zebra fish with wild zebra fish to obtain heterozygote with certain probability, collecting embryo, extracting genome, performing PCR with detection primer, cloning with TA, sequencing to determine genotype, and determining heritable F with effective mutation 1 Feeding zebra fish;
(5) after 3-4 months of sexual maturity, mutated F 1 And (3) cutting tails of adult male and female zebra fish, carrying out genotype identification and screening, and mating the mutant zebra fish again to obtain the homozygous hoxba gene cluster deletion mutant zebra fish.
Preferably, the zebra fish hoxba -/- The mutant is observed with pericardial cavity edema and abnormal cardiac development and malformation of cardiac cyclization at 3.5 dpf.
The invention also provides application of the zebra fish hoxba gene cluster deletion mutant as an animal model in researching biological functions of the hoxba gene cluster and diseases related to hoxba gene cluster deletion, wherein the zebra fish hoxba gene cluster deletion mutant is prepared by any one of the preparation methods of the zebra fish hoxba gene cluster deletion mutant.
Compared with the prior art, the invention has the following beneficial effects:
1) the invention designs gRNA targets on the 1 st exon of a hoxb13a gene and the 1 st exon of a hoxb1a gene respectively by using a CRISPR/Cas9 technology for the first time to delete large fragments of the hoxb gene cluster, wherein the hoxb gene cluster comprises 13 genes in total and has the full length of 116.9kb, and the hoxb1a, the hoxb2a, the hoxb3a, the hoxb4a, the hoxb5a, the hoxb6a, the hoxb7a, the hoxb8a, the hoxb9a, the hoxb10a, the hoxb11a, the hoxb12a and the hoxb13a are sequentially arranged from 3 'to 5', so that the specific knockout of the hoxb gene cluster is successfully realized, the zebra fish hoxb gene cluster deletion mutant is obtained, a method is provided for jointly knocking out a plurality of continuous genes on a genome and obtaining a stable mutant, and the phenotype of the hoxb gene cluster related to biological deletion of the hoxb gene cluster.
2) The hoxba gene cluster mutation can be stably inherited, and is convenient for the subsequent deep research on the functional mechanism of the hoxba gene cluster.
3)hoxba -/- The mutant zebra fish has obvious phenotype, and obvious cardiac development malformation phenomena such as pericardial cavity edema, abnormal cardiac cyclization and the like are observed at 3.5 dpf.
4) The invention realizes the site-specific knockout of the target gene by the knockout of large fragments of the genome without interfering the functions of other gene clusters, thereby providing a basis for subsequent related researches.
Drawings
FIG. 1 is a CRISPR/Cas 9-mediated deletion pattern of zebra fish hoxba gene cluster.
FIG. 2 shows the targeting sequence information and sites for the hoxba gene cluster deletion at the first exon of hoxb13a and the first exon of hoxb1a, respectively.
FIG. 3 shows the PCR detection of F with double outer primers in the process of mutant screening 0 ，F 1 ，F 2 Zebrafish genotype.
FIG. 4 is a graph of sequencing peaks showing successful deletion of the hoxba gene cluster in homozygous mutants.
FIG. 5 shows hoxba Gene cluster F 1 And (5) detecting the genotype of the mutant by PCR.
FIG. 6 is the gene sequence of homozygous mutant of the hoxba gene cluster.
FIG. 7 is a phenotypic map of mutations in zebrafish embryos following deletion of the hoxba gene cluster. Wild type (a) and hoxba mutant (b) cardiac phenotype at 3.5 dpf; (c) the heart cyclization angle of the hoxba mutant was significantly increased compared to the wild type. n is more than or equal to 3, and P is 0.00537. *: p <0.05, x: p <0.01, x: p < 0.001. Scale is 0.1 mm.
FIG. 8 is a phenotypic map of mutations in zebrafish embryos following deletion of the hoxba gene cluster. Heart phenotype of wild type (a) and hoxba mutant (d, g) at 3.5 dpf; the number indicates the location of the pericardial enlargement, (b, e, h) the heart region is enlarged and the black dashed line depicts the pericardial outline; (c, f, i) cardiac fluorescence labeling, cardiac cyclization of hoxba mutant was significantly abnormal compared to wild type, white dashed line delineating heart. The scale (a, d, g) is 0.2mm, and the scale (c, f, i) is 0.1 mm.
Detailed Description
The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.
Examples
1 Material and apparatus
1.1 Fish for experiments
The zebra fish used in the experiment is derived from a zebra fish platform of the aquatic product and life college of Shanghai ocean university.
1.2 plasmids
gRNA backbone plasmids were derived from literature: chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong JW, Xi JJ. genome editing with RNA-guided Cas9 nucleic in zebrafish embryo, Cell Res, 2013, 23(4): 465-.
1.3 Primary reagents
DNAClean&Contentrator-5(ZYMO RESEARCH，D4004)，
Figure BDA0003724216080000061
T7 in vitro Transcription Kit (Ambion, AM1314), ethanol (absolute ethanol) (national drug group chemical reagent Co., Ltd., 10009218), GenCrispr NLS-Cas9-NLS (Kinsry, Z03389-25), general DNA purification Kit (TIANGEN, DP204-03), Premix Taq TM (Ex Taq TM Version 2.0 plus dye)(TAKARA，RR902)，DNA Marker I(TIANGEN，MD101-02)，T7endonuclease 1(NEW ENGLAND
Figure BDA0003724216080000062
M0302L), DH5a competent cells (Tiangen Biotechnology Co., Ltd., CB101-03), 2BeasyTaq PCR Supermix (+ dye) (TAKARA, AS111-12), LB Broth (Shanghai Biolabs, D915KA6602), Rapid plasmid Mini kit (TIANGEN, DP105), LB Broth agar (Shanghai Biolabs, D911KA6566), pMD TM 19-T Vector Cloning Kit(TAKARA，6013)。
1.4 Main Instrument
PCR instrument (brand: BIO-RAD, model: c1000 Touch) TM Thermal Cycler), small centrifuge (brand: eppendorf, model: centrifuge 5424), shaking mixer (brand: VORTEX-GENIE, model: G560E), ultraviolet spectrophotometer (brand: thermo Scientific, model: nanodrop 2000C), autoclave (brand: SANYO, type: MLS-3780), electrophoresis apparatus (brand: BIO-RAD, type: PowerPac Basic), photographic glue apparatus (brand: Bio-Rad, model: gel Doc EZ Imager), electronic balance (brand: METTLER TOLEDO, type: AL104), Milli-Q Direct 8 ultra pure water system (brand: millipore, type: Milli-Q Direct 8), vertical pin puller (brand: NARISHIGE, model number: PC-10), constant temperature shaking table (brand: innova, type: 40R), needle grinder (brand: NARISHIGE, type: EG-400), micro-syringe pump (brand: WARNER, model: PLI-100A), thermostat water bath (brand: fine macro, type: h1401438, DK-8D), glassCapillary (brand: WPI, model: TW100F-4), 4 ℃ refrigerator (brand: Haier, model: HYC-610), 40 ℃ low temperature refrigerator (brand: Haier, model: DW-40L508), 80 ℃ ultra-low temperature refrigerator (brand: Pana-sonic, model: MDF-U53V).
2 method of experiment
2.1 design and screening of hoxba Gene knockout targets
(1) Specificity detection and selection analysis of hoxba Gene Cluster targets
a. Downloading sequence: the gene sequences of zebrafish hoxb13a and hoxb1a were looked up and downloaded in the Ensembl database.
b. Designing a target spot: designing targets on exon sequences behind hoxb13a and hoxb1a gene ATG by using http:// zifit.partners.org/ZiFiT/ChoiceMenu.aspx website to obtain two target sequences of hoxb13a, namely a phi GGATGAGCTGAAGAATATGG; ② GGACGTATCGACTTCAAGTT. Obtaining a sequence of three target points of hoxb1a, namely a phi GGAACTGGGACAACAAGTTA; GGTGCGTACTGCGGGGCACA; ③ GGCCTCTCAAGGAACGGAGA. Through experimental verification, a hoxb13a target point is found: GGATGAGCTGAAGAATATGG (SEQ ID NO:1), and hoxb1a target: GGAACTGGGACAACAAGTTA (SEQ ID NO:2) showed the best knock-out effect, and the specific target information is shown in Table 1.
Table 1: hoxb13a and hoxb1a target site sequences
Figure BDA0003724216080000071
c. And (3) target specificity detection: and (3) carrying out blast comparison on the designed target point sequence at the NCBI website to verify the target point specificity.
d. Parent detection: the WT zebra fish used for gene knockout is trimmed and subjected to alkaline lysis to obtain genome DNA, and a segment of sequence near a target point is amplified by PCR.
e. Sequencing and identifying: and (3) sequencing the PCR product, comparing a peak diagram with a sequence, and confirming that the parent is a homozygote without natural mutation, thereby ensuring that the subsequently prepared mutant is caused by gene knockout.
(2) Designing a target detection primer: the designed primers should be ensured to be more than 100bp away from both sides of the target, the difference between the distance from the upstream primer to the target and the distance from the downstream primer to the target should be more than 100bp and at least 50bp, the primer amplification should have specificity, the amplified fragment is about 500bp, and the primers are synthesized by Shanghai biological engineering Limited company (Table 2).
Table 2: primer information for experiments
Figure BDA0003724216080000072
Figure BDA0003724216080000081
(3) gRNA product synthesis: using the gRNA backbone plasmid as a template, primers T7-hoxb13a-sfd, T7-hoxb1a-sfd were used to amplify fragments with the gRNA reverse primer and 2 × EasyTaq PCR SuperMix (+ dye) respectively and purified with a kit.
(4) In vitro transcription: reaction system:
TABLE 3
Nuclease-free Water to 20μL
DNA template 1μg
10×Transcription Buffer 2μL
10mM ATP 1μL
10mM CTP 1μL
10mM GTP 1μL
10mM UTP 1μL
T7Enzyme Mix 2μL
Note that: finally, 10 × Transmission Buffer and T7Enzyme mix were added
Mixing, centrifuging for a short time, and incubating at 37 deg.C for 80 min; then, 1. mu.L of TURBO DNase was added to the system and mixed well, centrifuged briefly and incubated at 37 ℃ for 15 min.
(5) Purifying gRNA:
a. to 20. mu.L of the in vitro transcription system, 2.5. mu.L of 4M LiCl and 100. mu.L of body absolute ethanol were added, mixed well and centrifuged briefly and then placed in a freezer at-80 ℃ for at least 1 h.
b. After the time, the cells were removed from the freezer, centrifuged at 12000rmp at 4 ℃ for 15 min. The supernatant was discarded and the precipitate was washed with 70% ethanol. Centrifuge at 8000rmp for 5min at 4 ℃. After discarding the supernatant, the centrifuge tube was placed in a fume hood to volatilize the ethanol.
c. Adding appropriate amount of DEPC water to dissolve gRNA precipitate according to the size of the precipitate.
d. The concentration and OD values were measured by Nanodrop and electrophoresis.
Wherein the sequence of hoxb13a gRNA is
TAATACGACTCACTATAGGATGAGCTGAAGAATATGGGTTTTAGAGCTAGAAATAGCGGACAGATTCATGTCCTGGACGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO:6)；
The hoxb1a gRNA has the sequence
TAATACGACTCACTATAGGAACTGGGACAACAAGTTAGTTTTAGAGCTAGAAATAGCGGACAGATTCATGTCCTGGACGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT(SEQ ID NO:7)。
2.2 microinjection
The gRNA was mixed with Cas9 protein (purchased from gencriprpr NLS-Cas9-NLS (chryssper, Z03389-25)), and the mixed material was injected into zebrafish one-cell stage embryos using a microinjection instrument, leaving a batch of uninjected embryos as a control for each injection. Mixed injection final concentration: gRNA 100 ng/. mu.L, Cas9 protein 800 ng/. mu.L.
2.3 detection of successful knockout and knockout efficiency (PCR detection)
a. Extraction of fish egg genome
Each group of 5 eggs was incubated with 35. mu.L of 50mM NaOH at 95 ℃ for 20min, shaken, and centrifuged once. Then, 3.5. mu.L of 1M Tris-HCl (pH. apprxeq.8.0) was added thereto, followed by vigorous shaking and centrifugation.
b. PCR amplification of fragments of interest
And amplifying the target fragment according to the primer designed near the target point.
And (3) PCR reaction system:
TABLE 4
H 2 O to 25μL
Enzyme 12.5μL
F 0.5μL
R 0.5μL
Form panel 10ng
And (3) PCR reaction conditions:
pre-denaturation at 98 ℃ for 2 sec; denaturation at 98 ℃ for 10sec, annealing at 60.3 ℃ for 30sec, and extension at 72 ℃ for 1min for 32 cycles; further extension at 72 deg.C for 5 min; storing at 4 ℃.
2% agarose gel 120V electrophoresis for 25 min.
c. Electrophoretic detection
And (3) imaging the electrophoresed agarose gel by using a gel electrophoresis imager after electrophoresis, observing a target band, and judging whether the knockout is successful.
2.4 identification of genotype of mutant zebra fish with deletion homozygous for hoxba Gene Cluster
And carrying out genotype screening and identification on different deletion types.
3 results of the experiment
3.1 construction of mutant hoxba Gene Cluster
3.1.1 hoxbaF 0 Results of Gene knockout assay
The PCR result shows that the hoxba gene cluster is knocked out successfully. Sequencing peak plots showed that the genomic sequence between the hoxb13a gene target and the hoxb1a gene target was deleted, demonstrating successful knock-out (fig. 1, 2, 3, 4).
3.1.2 hoxbaF 0 germline transmission test result
Taking 20 tail hoxba F 0 Carrying out outcrossing on adult fish successfully knocked out by gene detection and wild zebra fish to obtain F 1 5 embryos are placed in one tube, 3-4 tubes are taken for double-lateral primer PCR identification, and the result shows that 1 zebra fish transmits mutation to offspring (figures 1, 2, 3 and 4).
3.1.3hoxba F 1 Heterozygous mutant zebra fish genotype identification
And (3) carrying out tail shearing detection on the hoxba genes of the zebra fish obtained by 71-tail outcrossing, carrying out PCR detection to obtain 48 positive zebra fish, and carrying out sequencing verification to confirm that large fragment deletion of hoxba gene clusters occurs (fig. 5 and 6).
3.1.4hoxba F 2 Mutant zebra fish phenotype observation photographing
(1) The hoxba gene cluster hybrid mutant was subjected to in vivo crossing, collected after egg laying and cultured for early embryo development observation, and significant cardiac malformations such as pericardial edema and abnormal cardiac cyclization were observed at 3.5dpf (fig. 7).
(2) To further characterize the hoxba gene cluster mutant, 3.5dpf hoxba gene cluster-deleted mutants and wild-type hearts were photographed for visualization and use in subsequent genotyping (FIG. 8).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Shanghai ocean university
Preparation method and application of <120> zebra fish hoxba gene cluster deletion mutant
<141> 2022-06-30
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ggaactggga caacaagtta 20
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taatacgact cactatagga tgagctgaag aatatgggtt ttagagctag aaatagc 57
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taatacgact cactatagga tgagctgaag aatatgggtt ttagagctag aaatagcgga 60
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taatacgact cactatagga actgggacaa caagttagtt ttagagctag aaatagcgga 60
cagattcatg tcctggacgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt 120
<210> 8
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cgtcacgtgg tactgctctc 20
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caatccacct gttttggggg 20
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acgctgatgg acgactttac g 21

Claims (9)

1. The preparation method of the zebra fish hoxba gene cluster deletion mutant is characterized by comprising the following steps of:
s1, determining a target point of hoxba gene cluster knockout, and designing gRNA sequences on the 1 st exon of the zebra fish hoxb13a gene and the 1 st exon of the hoxb1a gene respectively;
s2, designing an upstream primer T7-hoxb13a-sfd, a T7-hoxb1a-sfd and a downstream gRNA reverse primer of the synthetic gRNA;
s3, respectively carrying out PCR amplification by using upstream primers T7-hoxb13a-sfd, T7-hoxb1a-sfd and a gRNA reverse primer by using a gRNA framework plasmid as a template;
s4, carrying out in vitro transcription on the PCR product obtained in the step S3, and transforming to obtain hoxb13a gRNA and hoxb1a gRNA;
s5, mixing the hoxb13a gRNA and the hoxb1a gRNA with the Cas9 protein, and then performing microinjection and introduction into a zebra fish one-cell-stage embryo;
s6, culturing to obtain the stably inherited zebra fish hoxba gene cluster deletion mutant.
2. The method for preparing the zebra fish hoxba gene cluster deletion mutant according to claim 1, wherein in step S1, the gRNA sequence of the hoxb13a gene is shown as SEQ ID NO:1, and the gRNA sequence of the hoxb1a gene is shown as SEQ ID NO: 2.
3. The method for preparing the zebra fish hoxba gene cluster deletion mutant according to claim 1, wherein in step S2, sequences of T7-hoxb13a-sfd, T7-hoxb1a-sfd and gRNA reverse primers are shown as SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, respectively.
4. The method for preparing the zebra fish hoxba gene cluster deletion mutant according to claim 1, wherein in step S4, the sequence of hoxb13a gRNA is shown as SEQ ID NO. 6, and the sequence of hoxb1a gRNA is shown as SEQ ID NO. 7.
5. The method for preparing the zebra fish hoxba gene cluster deletion mutant according to claim 1, wherein in step S5, the final concentration of each of hoxb13a gRNA and hoxb1a gRNA is 100ng/μ L, the final concentration of Cas9 protein is 800ng/μ L, and the total volume is 1 nL.
6. The method for preparing the zebra fish hoxba gene cluster deletion mutant according to claim 1, wherein the step S6 specifically comprises the following steps:
a1, respectively taking zebra fish introduced with gRNA and Cas9 protein mixture and wild type uninjected zebra fish embryos for detecting hoxba gene cluster knockout efficiency, and determining F positive to hoxba gene cluster knockout 0 Culturing to adult fish;
a2, knocking out hoxba gene cluster to be positive F 0 Outcrossing adult fish and wild zebra fish, detecting heritability and effective mutation, screening heritability effective mutation F1, feeding to adult fish, and identifying genotype to obtain hoxba gene cluster F 1 Mutant zebrafish;
a3, cluster F of same mutated hoxba gene 1 Carrying out internal crossing on mutant zebra fish to obtain a hoxba gene cluster F 2 Mutant zebrafish;
a4, identified as F 2 Homozygotes of the hoxba gene cluster knockout in generations are deletion mutants of the stably inherited zebra fish hoxba gene cluster.
7. The method for preparing the zebra fish hoxba gene cluster deletion mutant as claimed in claim 6, wherein in step A1, the double outside primer PCR method is used to perform hoxba gene cluster knockout detection, and two pairs of detection primers hoxb13a-F/hoxb1a-R and hoxb1a-F/hoxb1a-R are used, wherein the sequences of the primers hoxb13a-F, hoxb1a-R and hoxb1a-F are shown as SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10 respectively.
8. The method for preparing the zebrafish hoxba gene cluster deletion mutant according to claim 6, wherein the zebrafish hoxba gene cluster deletion mutant is characterized in that the zebrafish hoxba gene cluster deletion mutant is a mutant of zebrafish hoxba -/- The mutant is observed with pericardial cavity edema and abnormal cardiac development and malformation of cardiac cyclization at 3.5 dpf.
9. Use of a zebrafish hoxba gene cluster deletion mutant as an animal model for studying the biological function of the hoxba gene cluster and diseases associated with hoxba gene cluster deletion, said zebrafish hoxba gene cluster deletion mutant being obtainable by the method of preparation of a zebrafish hoxba gene cluster deletion mutant according to any one of claims 1 to 8.
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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN109628454A (en) * 2019-01-30 2019-04-16 上海海洋大学 The construction method of zebra fish glycogen storage disease gys1 and gys2 gene mutation body

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Publication number Priority date Publication date Assignee Title
CN109628454A (en) * 2019-01-30 2019-04-16 上海海洋大学 The construction method of zebra fish glycogen storage disease gys1 and gys2 gene mutation body

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Title
KAZUYA YAMADA等: "An atlas of seven zebrafish hox cluster mutants provides insights into sub/neofunctionalization of vertebrate Hox clusters", 《DEVELOPMENT》, vol. 148, no. 11 *
刘保池主编: "《细胞治疗临床研究》", 复旦大学出版社, pages: 62 *
张绪帅: "CRISPR/Cas9系统介导的七鳃鳗和斑马鱼基因组编辑方法的建立与优化", 《中国优秀硕士学位论文全文数据库 基础科学辑》, pages 13 - 19 *
李佳: "CRISPR/Cas9技术在斑马鱼基因修饰中的应用", 《生命科学》, vol. 27, no. 1, 31 January 2015 (2015-01-31), pages 29 *
顾叶丹: "斑马鱼hoxb4a 在心脏发育中的功能研究", 《上海海洋大学学报》, 26 July 2021 (2021-07-26), pages 62 *

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