CN115851828A - Application of miRNA-140 and method for regulating development of craniofacial skeleton and scales of zebra fish - Google Patents

Abstract

The invention relates to the fields of life science and biotechnology, in particular to a method for constructing a zebra fish model based on miR-140 gene knockout, which can enable the zebra fish model to have craniofacial and squamosa dysplasia. By designing upstream and downstream targets of miR-140 gene, gene is knocked out by using CRISPR/Cas9 technology, so that a miR-140 gene knocked-out zebra fish model is constructed. Compared with the wild zebra fish of the AB strain, the miR-140 ^‑/‑ The shape of the scale at the back of the head of the zebra fish is changed, the scale is enlarged,Craniofacial bone dysplasia and tippy mouth indicate that zebra fish miR-140 regulates and controls the development of scales and craniofacial bones of zebra fish; the miR-140 gene effect can be further researched, and the method has great significance for the evolution research of scaled fishes and non-scaled fishes.

Description

The application of miRNA-140 and the method of regulating craniofacial skeleton and scale development in zebrafish

technical field

The invention belongs to the field of life science and biotechnology, and in particular relates to a method for constructing a zebrafish model based on miR-140 gene knockout to cause abnormal development of craniofacial and scales.

Background technique

MicroRNA is an endogenous non-coding RNA, which is known to control nearly 30% of gene expression in organisms. It activates target genes by complementary pairing with the 3' non-coding region (3'UTR) of the mRNA of the target gene The degradation of mRNA or the prevention of its translation into protein for gene regulation is a very important type of non-coding RNA in organisms. In recent years, the rise of CRISPR technology and as a powerful and convenient gene modification and editing technology has been sought after by many researchers, and it has been rapidly and widely used in human cells, fruit flies, mice, zebrafish and other organisms to study gene functions. Exploration and tumorigenesis research, etc. miR-140 is specifically highly expressed in chondrocytes, and miR-140 was first identified in zebrafish, and then found to be also specifically expressed in human and mouse cartilage. This gene exists on the short arm of chromosome 16 in humans and mice. In recent years, studies have found that it is involved in the differentiation of chondrocytes and is related to the pathogenesis of osteoarthritis. Miyaki et al. found that miR-140 ^-/- mice exhibited mild skeletal phenotypes, including short stature and light weight, as well as craniofacial abnormalities, short noses, and domed skulls. As the exoskeleton of fish, fish scales are homologous to teeth and bones. They are all derivatives of dermal skeleton. They can protect fish from external microorganisms, reduce friction damage caused by water when fish swim, and maintain Important biological functions such as the special body shape of fish. At present, the research on zebrafish scales is mainly focused on the formation mode and morphogenesis. At the molecular level, it is mainly to knock out EDA, EDAR, FGFRs and other genes related to fish scales, but the miRNAs involved in scale development are not studied. There is no relevant literature report, and its research is also of great significance for revealing the evolution of scaled and scaleless fishes.

Contents of the invention

The present invention aims to provide the application of miR-140 gene.

The present invention provides a method for knocking out zebrafish miR-140.

The purpose of the present invention is also to use zebrafish as a model to knock out miR-140 through CRISPR/Cas9 technology, thereby simulating its influence on craniofacial bone and scale development in fish.

The invention also provides a method for regulating craniofacial bone development or scale development of zebrafish.

The technical solution of the present invention is the application of miRNA-140 in regulating craniofacial bone development or scale development of zebrafish.

By knocking out the miRNA gene, the miR-140 gene knockout zebrafish model was constructed, and then its function was observed and verified by methods such as calcein staining and micro-ct. Through repeated comparison and observation, it was found that miR-140 ^-/- zebrafish Compared with the wild zebrafish of the AB strain, the shape of the scales at the back of the head changed, the scales became larger, the craniofacial bones developed abnormally, and the mouth became sharper, indicating that zebrafish miR-140 can regulate the development of its scales and craniofacial bones.

A method for regulating craniofacial bone development or scale development of zebrafish, specifically, knocking out the miRNA-140 gene of the zebrafish or enhancing the expression of the miRNA-140 gene.

After knocking out the miRNA-140 gene in zebrafish, the shape of the scales at the back of the head of the zebrafish changed, the scales became larger, the craniofacial bones developed abnormally, and the mouth became pointed. Enhancing the expression of zebrafish miRNA-140 gene, a method for constructing a zebrafish model with abnormal craniofacial and scale development, specifically, knocking out the miRNA-140 gene of zebrafish. Specific steps include:

(1) Knock out the miRNA-140 gene in the one-cell stage fertilized eggs of zebrafish, and obtain the F0 generation of zebrafish heterozygote with miR-140 gene knockout;

(2) The F0 generation was crossed with the AB wild zebrafish to obtain the F1 generation, and a heterozygote for the deletion of the miR-140 gene was obtained through gene sequencing;

(3) Incross the mature F1 generation of miR-140 gene-deficient female and male heterozygotes to obtain the F2 generation, and obtain F2 generation of male and female homozygous zebrafish with miR-140 gene deletion.

Preferably, step (4) is also included: mating the male and female homozygous zebrafish of the F2 generation with miR-140 gene deletion to generate the F3 generation, homozygous zebrafish.

Through the above method, a zebrafish model with abnormal craniofacial bone development and enlarged scales can be established.

The method for knocking out the zebrafish miRNA-140 gene is specifically to use CRISPR/Cas9 technology to double knock out miR-140.

Knocking out the 297bp nucleotide sequence of No. 217-513 in SEQ ID No.1 includes the following steps: injecting Cas9 and upstream and downstream gRNAs into zebrafish one-cell stage fertilized eggs.

The upstream and downstream gRNA is prepared by the following steps:

(A) Using the oligonucleotide sequences shown in SEQ ID No.6 and SEQ ID No.7, and the sgRNA backbone sequence shown in SEQ ID No.6 as templates, amplify and obtain gRNA in vitro transcription templates;

(B) According to the gRNA in vitro transcription template, perform in vitro transcription of the T7 promoter to obtain upstream and downstream gRNAs.

Oligonucleotides include on-target oligonucleotides and on-target oligonucleotides, including the T7 promoter, target sequence, and sequence that recognizes the sgRNA backbone.

Target oligonucleotide Oligo-1:

GATCACTAATACGACTCACTATAGGAGAGGAAGGCCTGGATTAGTTTTTAGAGCTAGAAATAGC (SEQ ID No. 6).

Next target oligonucleotide Oligo-2:

GATCACTAATACGACTCACTATAGGCAAAGATGATTGAGGGTGGTTTTTAGAGCTAGAAATAGC (SEQ ID No. 7)

Wherein GATCACTAATACGACTCACTATA is T7 promoter, and GGAGAGGAAGGCCTGGATTA and GGCAAAGATGATTGAGGGTG in SEQ ID No.6 and SEQ ID No.7 are target sequences respectively. GTTTTAGAGCTAGAAATAGC is used to identify the tethered sgRNA backbone.

sgRNA-scaffold:

AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC (SEQ ID No. 8).

Knockout was performed by targeting SEQ ID NO.2 and SEQ ID NO.3 located upstream and downstream of the miR-140 gene.

Upper target TS-140-1: 5'-GGAGAGGAAGGCCTGGATTA-3' (SEQ ID NO.2)

Lower target TS-140-2: 5'-GGCAAAGATGATTGAGGGTG-3' (SEQ ID NO.3).

A primer for identifying the phenotype of the zebrafish miRNA-140 gene, the upstream and downstream primer sequences of which are shown in SEQ ID No.4 and SEQ ID No.5.

TS-140-F: 5'-TGGGAATGCATTATTTGCTGATTTG-3' (SEQ ID NO.4)

TS-140-R: 5'-ATCGGGGCCGTAACTCTAAG-3' (SEQ ID NO.5)

The present invention mainly designs the upstream and downstream targets of the miR-140 gene, and uses CRISPR/Cas9 technology to double-knock miR-140, thereby constructing the miR-140 gene knockout zebrafish model. Using calcein staining and micro-ct to observe and verify its function, it was found that compared with the wild zebrafish of the AB strain, miR-140 ^-/- zebrafish had changes in the shape of scales at the back of the head, larger scales, and craniofacial bone development Abnormal, pointed mouth, indicating that zebrafish miR-140 regulates the development of its scales and craniofacial bones.

The present invention discovers the regulatory relationship between the zebrafish miR-140 gene and the development of scales and craniofacial bones, and realizes the knockout of the miR-140 gene through the upstream and downstream targets of the miR-140 gene, and constructs miR-140 The gene knockout zebrafish model can further study the role of miR-140, and it is also of great significance for the evolution of scaled and scaleless fishes.

Description of drawings

Figure 1 is a schematic diagram of the sequence of miRNA-140, upstream and downstream targets, and identification primers of SEQ ID NO.1.

Figure 2 is the sequencing map of the zebrafish gene miR-140 "-297bp" type

Figure 3 is the phenotype of miR-140 ^-/- and wild-type zebrafish in the F3 generation

Figure 4 is a side view of miR-140 ^-/- and wild-type zebrafish micro-ct in the F3 generation

Detailed ways

Example 1

Get the zebrafish dre-miR-140 sequence and the complete sequence within 500bp upstream and 1000bp downstream from Ensembl (http://asia.ensembl.org/index.html), miRbase (http://www.mirbase.org/ ) database to query its precursor and mature sequences, and the NCBI (http://www.ncbi.nlm.nih.gov/) website will further verify and supplement its information.

Based on the ZiFiT (http://zifit.partners.org/ZiFiT/) online software, design the upstream and downstream gRNA target TS-140-1/2 (Targetsite sequences) target sites for zebrafish miR-140 gene knockout, and synthesize Oligo and sgRNA-scaffold, according to the template to obtain gRNA in vitro transcription template by PCR, and then perform in vitro transcription of T7 promoter according to the in vitro transcription template; and carry out miR-140 gene knockout experiment on AB type wild zebrafish embryos.

1. Screening and preparation of available gRNA targets for zebrafish miR-140 knockout

The position of the miR-140 gene is the 372-476th position of the sequence of SEQ ID No.1, as shown in Figure 1, it is a bold, slanted and underlined sequence in SEQ ID NO.1, a total of 105bp, which is the zebrafish miR -140 gene, which is located in the 16th intron region of the wwp2 gene.

In addition, the bold 20bp sequence in Figure 1 is the upstream and downstream targets, designed in the WWP2 gene. The sequences underlined with wavy lines are identification primers, starting from the bolded and underlined upper target sequence to the bolded and underlined lower target sequence, the middle 297bp sequence (the gray part in Figure 1) was detected by sequencing. Knock it off completely. The designed target site sequence is as follows:

Upper target TS-140-1: 5'-GGAGAGGAAGGCCTGGATTA-3' (SEQ ID NO.2), lower target TS-140-2: 5'-GGCAAAGATGATTGAGGGTG-3' (SEQ ID NO.3), which is Reverse target, corresponding to (caccctc aatcatctttgcc ) of the above sequence.

Design the upper target oligonucleotide and the upper target oligonucleotide, the sequences of which are shown in SEQ ID No.6 and SEQ ID No.7 respectively, including the T7 promoter, target sequence and sequence for recognizing the sgRNA backbone.

Target oligonucleotide Oligo-1:

GATCACTAATACGACTCACTATAGGAGAGGAAGGCCTGGATTAGTTTTTAGAGCTAGAAATAGC (SEQ ID No. 6).

Next target oligonucleotide Oligo-2:

GATCACTAATACGACTCACTATAGGCAAAGATGATTGAGGGTGGTTTTAGAGCTAGAAATAGC (SEQ ID No. 7).

The sgRNA backbone (sgRNA-scaffold) sequence is shown in SEQ ID No.8:

AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC (SEQ ID No. 8)

Use the Primer blast on the NCBI website to design the identification primers online, and the selection conditions are: (1) the distance between the upstream and downstream primers is 100 bp from the target; (2) the length of the upstream and downstream primers is 20-23 bp; (3) the amplified fragment is about 500 bp; ( 4) The designed primers have high specificity (that is, only the required bands can be amplified). The designed identification primers are as follows:

TS-140-F: 5'-TGGGAATGCATTATTTGCTGATTTG-3' (SEQ ID NO.4)

TS-140-R: 5'-ATCGGGGCCGTAACTCTAAG-3' (SEQ ID NO.5)

The size of the fragment amplified by the primers can be used to distinguish the following three phenotypes: the result of the wild type is 695bp; the gel map of the heterozygote has a band of 695bp and a band of 398bp below it; the homozygote only has a band of 398bp The size of the strip.

2. Synthesis and purification of gRNA in vitro transcription template:

Synthetic Oligo, sgRNA-scaffold and identification primers were all dissolved in sterilized ddH ₂ O to 10 μM.

The 40 μL system is: Oligo (10 μM) 3 μL, SgRNA-scaffold (10 μM) 3 μL, 2xEasyTaq PCRSuperMix (+dye) 20 μL, add water to 40 μL. In addition, the PCR reaction was 94°C for 3min, 94°C for 30s, 65°C for 30s, 72°C for 1min, 72°C for 5min, 4°C∞, 34 cycles. A total of 160 μL of 4 tubes was synthesized into one tube for column purification. For specific steps, refer to the instructions of the QIAquick PCR purification kit kit from QIAGEN. After purification, use NanoDrop (Thermo, USA) to measure its concentration, and run the gel at 160V/22min to check whether the size is correct.

3. In vitro transcription and purification of gRNA in vitro transcription template:

The present invention utilizes the T7 promoter for in vitro transcription. For specific operation steps, refer to the mMESSAGE mMACHINE T7 Ultra Kit kit from Life Company for in vitro transcription, and complete the in vitro transcription according to the instructions.

The specific steps for purifying gRNA are as follows:

First, add 1 μL TURBO DNase to the above transcription product, incubate at 37°C for 15 minutes to remove excess untranscribed template DNA, and then add 1 μL 0.5M EDTA to terminate the reaction. Then add 2.5 μL of 4M LiCl and 100 μL of absolute ethanol, mix thoroughly, and store at -80°C overnight. The next day, centrifuge at -4°C at 12,000 rpm for 15 min, discard the supernatant, add 1 mL of 70% ice ethanol to wash, and finally add Nuclease-free water to resuspend RNA, use NanoDrop to determine the concentration, and electrophoresis to detect the size and quality.

Example 2 Construction of a zebrafish model for knocking out the miRNA-140 gene

1. Microinjection

Cas9 protein (purchased directly from Genscript Company) and the upstream and downstream transcribed gRNA obtained in Example 1 were co-injected into zebrafish one-cell stage fertilized eggs, and the dose of mixed injected Cas9 protein and gRNA: 400pg/μL: 100pg/ μL, injection volume: 1nL/zygote. When injecting, place the target on ice and mix it immediately.

Specifically, the above-mentioned process of obtaining stable inherited miR-140 gene knockout homozygous zebrafish is as follows: (1) Perform gene sequencing on the injected zebrafish to obtain miR-140 gene knockout zebrafish heterozygous F0 generation ; (2) The F1 generation was obtained by crossing the F0 generation with the AB-type wild zebrafish, and the "-297bp" (deletion of 297bp) heterozygote was obtained through gene sequencing; (3) The mature F1 generation "-297bp" (deletion of 297bp) type Female and male heterozygotes were inbred to obtain the F2 generation, and the "-297bp" (deletion of 297bp) gene-edited F2 generation was obtained after gene sequencing. Homozygous male and female. (4) Mating "-297bp" (deletion of 297bp) gene-edited F2 generation female homozygous zebrafish with "-297bp" (deletion of 297bp) gene-editing F2 generation male heterozygote zebrafish to generate F3 generation, and obtained F3 after gene sequencing "-297bp" (deletion of 297bp) type female and male homozygous zebrafish. Among them, the upper targets are TS-140-1: 5'-GGAGAGGAAGGCCTGGATTA-3' (SEQ ID NO.2) and TS-140-2: 5'-GGCAAAGATGATTGAGGGTG-3' (SEQ ID NO.3). The primers used for gene sequencing were TS-140-F: 5'-TGGGAATGCATTATTTGCTGATTTG-3' (SEQ ID NO. 4) and TS-140-R: 5'-ATCGGGGCCGTAACTCTAAG-3' (SEQ ID NO. 5).

2. Detection of the effectiveness of F0 embryo injection:

After the injection, the zebrafish embryos developed to 24h-48h, and ten groups (5 eggs/group) were randomly selected, and the genomic DNA was crudely extracted by alkaline cleavage. The main operation steps were as follows:

(1) Add 50 μL of 50 mM NaoH solution to each tube of zebrafish embryos and incubate at 95 °C for 10 min;

(2) Briefly centrifuge to bring the liquid to the bottom of the tube, vortex for about 1 min, and then incubate at 95°C for 10 min;

(3) After short centrifugation, place on ice for 1-2 minutes, add 5 μL Tri-HCl (PH=8.0), vortex, centrifuge at 12,000 rpm for 10 minutes at room temperature, store at 4°C for short-term storage, and -20°C for long-term storage ;

(4) Finally, a PCR reaction was performed using the extracted zebrafish genomic DNA as a template to identify the effectiveness of the zebrafish knockout.

The identified primer sequences are as follows:

TS-140-F: 5'-TGGGAATGCATTATTTGCTGATTTG-3' (SEQ ID NO.4)

TS-140-R: 5'-ATCGGGGCCGTAACTCTAAG-3' (SEQ ID NO.5)

The 20 μL system is: 1 μL of TS-140-F (10 μM), 1 μL of TS-140-R (10 μM), 10 μL of 2xEasyTaq PCR SuperMix (+dye), 1 μL of the extracted genome, and finally add water to 20 μL. In addition, the PCR reaction was 94°C for 3min, 94°C for 30s, 60°C for 30s, 72°C for 1min, 72°C for 5min, 4°C∞, 34 cycles.

Electrophoresis was run to test the effectiveness of the knockout, and the PCR products of possible mutants were sequenced in one direction. The peak diagram was viewed using the software Chrome, and the blast n of the NCBI website was used to compare the sequences. It was found that compared with the wild type, the mutant had double peaks near the target point, indicating the effectiveness of the knockout. The rest of the fertilized eggs were raised to 2 months old, and the tail was cut according to the above-mentioned alkaline cracking method. Extract genomic DNA, perform PCR reaction with the 20 μL system mentioned above, run the gel and send it for testing. After identification, mutant heterozygotes (-297bp) were mated with AB wild-type zebrafish to obtain the F1 generation.

3. Screening of F1 generation mutants

The mutants selected from the F0 generation and the F1 generation zebrafish obtained from the AB wild-type zebrafish were raised until they could lay eggs, and the tail was cut to screen the mutants in the same way as above. After gene sequencing, "-297bp" (deletion of 297bp ) heterozygotes, and then the screened male and female mutants were mated to obtain the F2 generation.

As shown in the sequence comparison diagram in Figure 2, it can be clearly seen that the homozygous sequencing starts from GGCCTGGATTA in the upper target 5'-GGAGAGGAAGGCCTGGATTA-3' and ends with CACCCTC in the lower target CACCCTC AATCATCTTTGCC . Among them, the WT sequence lacks 297 bp of the sequence in the period, and the sequence in the period is omitted with "-" because of too many sequences.

In addition, comparing the peak diagrams of the two (the position shown by the arrow mark is the position where the target sequence begins, and the underline is the first 7 bases of the target sequence), it is obvious that the 7 sequences before the sequence of the homozygous F2 generation are On the target sequence, but the wild type is not.

4. Screening of homozygous F2 generation and obtaining of F3 generation

Using the same method as above for tail-cutting screening, after gene sequencing, the F2 generation homozygous male and female adults of the "-297bp" (297bp deletion) gene were obtained, and the F3 generation was obtained by mating.

Example 3 Calcein staining to detect the phenotypic detection of F3 generation homozygotes

The F3 generation zebrafish obtained in Example 2 was used for phenotypic detection.

First, anesthetize the zebrafish with 1% tricaine, and after complete anesthesia, place them in a 2% calcein solution prepared in fish water for 3 to 5 minutes in the dark, then put them back into the fish water, and remove them by changing the water. The excess dye solution was anesthetized before taking pictures, and then the shape of the scales was observed under a fluorescent microscope. From Figure 3, we can clearly see that miR-140 ^-/- compared with WT wild-type zebrafish, the craniofacial bone changes, the mouth becomes sharper, the scales at the back of the head become larger, and the shape changes.

The micro-ct of embodiment 4 homozygote

After the F3 generation homozygous body was fixed with 4% paraformaldehyde, it was sent to Pingsheng Medical Biotechnology Co., Ltd. for micro-ct. From Figure 4, we can clearly see that the craniofacial bone of miR-140 ^-/- zebrafish has changed significantly, which further shows that miR-140 plays a very important role in the regulation of the craniofacial bone of zebrafish.

The above examples are only preferred examples of the present invention, and do not limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included within the protection scope of the present invention .

sequence listing

<120> The application of miRNA-140 and the method to regulate the development of craniofacial skeleton and scales in zebrafish

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 695

<212>DNA

<213> Zebrafish (Danio rerio)

<400> 1

tgggaatgca ttatttgctg atttgtatta tgaaacaaat tgataattat ttttatttga 60

tcttttgaca tgtacaaaca tcgccgtgca acatatccat attgttcatg taatccacta 120

atataccctc aacaccgtcc cgtttctcct cagatcatga acatgaagcc gtatgacctc 180

cgccggcgac tctacataat catgagagga gaggaaggcc tggattacgg aggaatcgcc 240

aggcaagtca aaccctgtag catcccgttg tccgtcatcc tggtgctcct gttcagtgtt 300

tgttgtttgg ggagttctgt ggtgttcctc tgcctgtctg ctggagctca tgtagttgtc 360

ttcctgtgtc tgtgtttgtc tcctgtgtcc cgtcagtggt tttaccctat ggtaggttac 420

gtcatgctgt tctaccacag ggtagaacca cggacgggat gtctggaggt gtctgcgtcg 480

ccctgtggtg ccaaaataaa caccctcacc ctcaatcatc tttgcccctt taccttctgt 540

cccgtcgggc tccgagatct tgggttctgc tgctggaccg gttttgattt tcgacgtgtc 600

tgtgtttgtg tttttgttca gtctgtctgc atcttcatca ttgtgtatgg cacttttgtg 660

gatttcttta cagatcttag agttacggcc ccgat 695

<210> 2

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 2

ggagaggaag gcctggatta 20

<210> 3

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 3

ggcaaagatg attgagggtg 20

<210> 4

<211> 25

<212>DNA

<213> Artificial Sequence

<400> 4

tgggaatgca ttatttgctg atttg 25

<210> 5

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 5

atcggggccg taactctaag 20

<210> 6

<211> 63

<212>DNA

<213> Artificial Sequence

<400> 6

gatcactaat acgactcact ataggagagg aaggcctgga ttagttttag agctagaaat 60

agc 63

<210> 7

<211> 63

<212>DNA

<213> Artificial Sequence

<400> 7

gatcactaat acgactcact ataggcaaag atgattgagg gtggttttag agctagaaat 60

agc 63

<210> 8

<211> 80

<212>DNA

<213> Artificial Sequence

<400> 8

aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt 60

gctatttcta gctctaaaac 80

Claims (8)

1. Application of miRNA-140 in regulation and control of development of craniofacial bones or scales of zebra fish.
2. 2. A method for regulating and controlling development or scale development of a craniofacial bone of zebra fish is characterized in that miRNA-140 genes of the zebra fish are knocked out, expression of the miRNA-140 genes is reduced, or expression of the miRNA-140 genes is enhanced.
3. 3. The method for constructing the zebra fish model with craniofacial and squamosa dysplasia is characterized in that miRNA-140 genes of the zebra fish are knocked out.
4. 4. The method of constructing a craniofacial and squamosal dysplastic zebrafish model according to claim 4, wherein the steps comprise:

   (1) Knocking out miRNA-40 genes in fertilized eggs of zebra fish in a cell stage to obtain a zebra fish heterozygote F0 generation with miR-140 gene knocked out;

   (2) Hybridizing the F0 generation with AB type wild zebra fish to obtain an F1 generation, and performing gene sequencing to obtain a miR-140 gene deletion heterozygote;

   (3) Carrying out internal hybridization on mature F1 generation miR-140 gene deletion type male and female heterozygotes to obtain an F2 generation, and obtaining F2 generation miR-140 gene deletion male and female homozygote zebra fish.
5. 5. The method for constructing a craniofacial and squamosal dysplastic zebrafish model according to claim 4, further comprising the step (4): and (3) mating the male and female homozygote zebra fish with the miR-140 gene deleted in the F2 generation to generate the F3 generation homozygote zebra fish.
6. 6. The method for knocking out the zebra fish miRNA-140 gene is characterized in that a nucleotide sequence with the length of 297bp from 217 to 513 in SEQ ID No.1 is knocked out, and comprises the following steps: cas9 and upstream and downstream grnas were injected into one-cell stage zygotes of zebrafish.
7. 7. The method for knocking out zebrafish miRNA-140 gene according to claim 6, wherein the upstream and downstream gRNAs are prepared by the following steps:

   (A) Taking oligonucleotide sequences shown as SEQ ID No.6 and SEQ ID No.7 and sgRNA framework sequences as templates, and amplifying to obtain a gRNA in-vitro transcription template;

   (B) And (3) carrying out in-vitro transcription of the T7 promoter according to a gRNA in-vitro transcription template to obtain upstream and downstream gRNAs.
8. 8. The primer for identifying the phenotype of the zebra fish miRNA-140 gene is characterized in that the sequences of the upstream primer and the downstream primer are shown as SEQ ID No.4 and SEQ ID No. 5.

Images