A method for preparing zebrafish trmt61a maternal zygotic mutant

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to a zebrafish trmt61a The method for preparing maternal zygotic mutants comprises the following steps: targeting zebrafish trmt61a The sgRNA and Cas9 protein of the gene were co-injected into one-cell embryos of wild-type zebrafish. trmt61a Heterozygous mutant; construct pGGDestISceIEG‑4sgRNA plasmid and co-inject it with endonuclease and reaction buffer into Tg( zpc :zcas9) transgenic zebrafish one-cell embryos to obtain adult zebrafish F0 generation; the female fish of the adult zebrafish F0 generation were crossed with trmt61a Heterozygous mutant male fish were crossed to obtain zebrafish trmt61a Maternal zygotic mutants. This method breaks through trmt61a The limitation of homozygous gene mutation causing lethality enables the rapid preparation of maternal zygotic mutants.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a zebrafish trmt61a Method for preparing maternal zygotic mutants. Background Art

[0002] tRNA m 1 A is a tRNA methylation modification, m 1 A (N1-methyladenosine) is a common modification on tRNA that regulates gene expression at multiple levels. 1 A-modified tRNA has higher structural stability and can be preferentially recruited into active polymers to promote translation. 1 A58-modified methyltransferase complex, in which Trmt61a is an enzyme belonging to the tRNA methyltransferase family. It is the subunit of the enzyme catalytic activity in the complex and is mainly expressed in the cell nucleus.

[0003] As a widely used model animal, zebrafish has a high degree of homology with human genes and has outstanding advantages as a model organism, which means that its experimental results are applicable to humans in most cases. At the same time, it has many characteristics such as strong reproductive ability, in vitro fertilization and development, transparent embryos, short sexual maturity cycle, small size and easy breeding. Therefore, it plays an important role in embryonic development, drug screening and disease research.

[0004] In zebrafish, there are more than 16,000 genes with significant maternal input, accounting for 66% of the total number of zebrafish genes. In order to study the function of these genes, it is necessary not only to eliminate zygotic gene expression, but also to inhibit its maternal product, that is, to rely on obtaining maternal zygotic mutants. However, if some gene mutations lead to homozygous lethality or infertility, it will be impossible to obtain maternal zygotic mutants through classical genetic methods. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the object of the present invention is to provide a zebrafish trmt61a Method for preparing maternal zygotic mutants.

[0006] In order to achieve this purpose, the technical solution adopted by the present invention is:

[0007] A zebrafish trmt61a The method for preparing a maternal zygotic mutant comprises the following steps:

[0008] S100. Targeting zebrafish trmt61a The sgRNA and Cas9 protein of the gene were co-injected into one-cell embryos of wild-type zebrafish and cultured to adulthood. trmt61a Heterozygous mutants;

[0009] S200. Construct pGGDestISceIEG-4sgRNA plasmid and inject it together with endonuclease and reaction buffer into Tg( zpc :zcas9) zebrafish one-cell embryos were cultured to adulthood to obtain adult zebrafish Tg( zpc : zcas9; U6 :4sgRNA) F0 generation;

[0010] S300. Make adult zebrafish Tg( zpc : zcas9; U6 :4sgRNA) F0 generation female fish and trmt61a Heterozygous mutant male fish were crossed, identified, and selected to obtain zebrafish trmt61a Maternal zygotic mutants.

[0011] Furthermore, targeting zebrafish trmt61a The site of the gene's sgRNA is located in the second exon region of chromosome 13.

[0012] Furthermore, the sgRNA in step S100 is sgRNA0, whose nucleotide sequence is shown in sequence 1. The pGGDestISceIEG-4sgRNA plasmid in step S200 includes four sgRNAs, namely sgRNA1, sgRNA2, sgRNA3 and sgRNA4. The nucleotide sequence of sgRNA1 is shown in sequence 2, the nucleotide sequence of sgRNA2 is shown in sequence 3, the nucleotide sequence of sgRNA3 is shown in sequence 4, and the nucleotide sequence of sgRNA4 is shown in sequence 5.

[0013] Furthermore, in step S100, the preparation process of sgRNA is as follows:

[0014] S110. Using the pMD19-sgRNA scaffold plasmid as a template, PCR amplification was performed using upstream primers and downstream primers to obtain the DNA template required for synthesizing gRNA;

[0015] S120. Generate sgRNA by in vitro transcription using DNA template.

[0016] Further, the construction of pGGDestISceIEG-4sgRNA plasmid in S200 includes the following steps:

[0017] S210. Construction of U6-sgRNA1-gRNA scaffold fragment;

[0018] S220. Construction of U6-sgRNA2-gRNA scaffold fragment;

[0019] S230. Construction of U6-sgRNA3-gRNA scaffold fragment;

[0020] S240. Construction of U6-sgRNA4-gRNA scaffold fragment;

[0021] S250. The constructed U6-sgRNA1-gRNA scaffold fragment, U6-sgRNA2-gRNA scaffold fragment, U6-sgRNA3-gRNA scaffold fragment and U6-sgRNA4-gRNA scaffold fragment were assembled with the pGGDestISceIEG vector fragment to obtain the pGGDestISceIEG-4sgRNA plasmid.

[0022] Furthermore, the construction of the U6-sgRNA1-gRNA scaffold fragment in S210 includes the following steps:

[0023] S211. Using pU6sgRNA plasmid as template and sequence 6 and sequence 7 as primers, PCR was used to clone U6 to obtain U6-sgRNA1 fragment;

[0024] S212. Using pU6sgRNA plasmid as a template and sequence 8 and sequence 9 as primers, the gRNA scaffold was cloned by PCR to obtain the sgRNA1-gRNA scaffold fragment;

[0025] S213. Using the mixture of U6-sgRNA1 fragment and sgRNA1-gRNA scaffold fragment as template and sequence 6 and sequence 9 primers, overlap extension PCR was performed to obtain U6-sgRNA1-gRNA scaffold fragment.

[0026] Furthermore, the construction of the U6-sgRNA2-gRNA scaffold fragment in S220 includes the following steps:

[0027] S221. Using pU6sgRNA plasmid as template and sequence 10 and sequence 11 as primers, PCR was used to clone U6 to prepare U6-sgRNA2 fragment;

[0028] S222. Using pU6sgRNA plasmid as a template and sequence 12 and sequence 13 as primers, the gRNA scaffold was cloned by PCR to obtain the sgRNA2-gRNA scaffold fragment;

[0029] S223. Using a mixture of the U6-sgRNA2 fragment and the sgRNA2-gRNA scaffold fragment as a template and sequence 10 and sequence 13 as primers, overlap extension PCR was performed to obtain the U6-sgRNA2-gRNA scaffold fragment.

[0030] Furthermore, the construction of the U6-sgRNA3-gRNA scaffold fragment in S230 includes the following steps:

[0031] S231. Using pU6sgRNA plasmid as template and sequence 14 and sequence 15 as primers, PCR was used to clone U6 to prepare U6-sgRNA3 fragment;

[0032] S232. Using pU6sgRNA plasmid as template and sequence 16 and sequence 17 as primers, the gRNA scaffold was cloned by PCR to obtain the sgRNA3-gRNA scaffold fragment;

[0033] S233. Using a mixture of the U6-sgRNA3 fragment and the sgRNA3-gRNA scaffold fragment as a template and sequence 14 and sequence 17 as primers, overlap extension PCR was performed to obtain the U6-sgRNA3-gRNA scaffold fragment.

[0034] Furthermore, the construction of the U6-sgRNA4-gRNA scaffold fragment in S240 includes the following steps:

[0035] S241. Using pU6sgRNA plasmid as template and sequence 18 and sequence 19 as primers, PCR was used to clone U6 to prepare U6-sgRNA4 fragment;

[0036] S242. Using pU6sgRNA plasmid as a template and sequence 20 and sequence 21 as primers, the gRNA scaffold was cloned by PCR to obtain the sgRNA4-gRNA scaffold fragment;

[0037] S243. Using a mixture of the U6-sgRNA4 fragment and the sgRNA4-gRNA scaffold fragment as a template and sequence 18 and sequence 21 as primers, overlap extension PCR was performed to obtain the U6-sgRNA4-gRNA scaffold fragment.

[0038] Furthermore, in step S100, the mass ratio of the added amounts of sgRNA and Cas9 is 2:3 to 3:2.

[0039] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0040] A zebrafish provided by the present invention trmt61a Method for preparing maternal zygotic mutants, constructed using CRISPR / Cas9 technology trmt61a Heterozygous mutants, combined with endonuclease-mediated transgenic zebrafish construction technology, achieved knockout in zebrafish oocytes trmt61a Gene expression in oocytes trmt61a The removal of mRNA and Trmt61a protein was achieved by trmt61a Heterozygous mutant hybridization, a breakthrough trmt61a The limitation of homozygous lethal mutations in genes was quickly obtained. trmt61a Maternal zygotic mutants.

[0041] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The embodiment 1 of the present invention provides trmt61a mRNA expression in wild-type zebrafish embryos at different stages.

[0043] Figure 2 The sgRNA sequence provided in Example 2 of the present invention is trmt61a Schematic diagram of the site on the gene.

[0044] Figure 3 Embodiment 2 of the present invention provides trmt61a Figure 2. Sequencing results of heterozygous mutants.

[0045] Figure 4 This is the sequencing result after the high-efficiency sgRNA injection provided in Example 2 of the present invention.

[0046] Figure 5 The zebrafish at 36 hpf provided in Example 2 of the present invention trmt61a Western blotting of the expression of heterozygous mutant Trmt61a protein.

[0047] Figure 6 This is the pGGDestISceIEG-4sgRNA (trmt61a) plasmid map provided in Example 4 of the present invention.

[0048] Figure 7 The transgenic zebrafish Tg( zpc : zcas9; U6:4sgRNA) F1 generation fluorescence photograph.

[0049] Figure 8 Embodiment 5 of the present invention provides trmt61a Figure 2. Sequencing results of maternal zygotic mutants.

[0050] Figure 9 The detection method provided in Example 5 of the present invention trmt61a Maternal zygotic mutants trmt61a mRNA levels.

[0051] Figure 10 Embodiment 5 of the present invention provides trmt61a Immunofluorescence images of maternal zygotic mutants at the 256-cell stage.

[0052] Among them: Sibling is a sibling control; heter Z trmt61a for trmt61a Heterozygous mutant; heterZ trmt61a (maternal factor cleared) trmt61a Heterozygous mutant; homoZ trmt61a for trmt61a Homozygous mutant; M trmt61a for trmt61a Maternal zygotic mutants. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0054] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0055]

[0056]

[0057] The preparation method provided by the present invention is designed and synthesized for trmt61a Gene-specific sgRNA was co-injected into wild-type zebrafish embryos at the 1-cell stage and obtained through identification, screening and culture. trmt61a Heterozygous mutants. Four mutants targeting trmt61a Gene-specific efficient sgRNA, a fragment containing U6 promoter sequence, sgRNA target sequence and gRNA scaffold sequence was amplified, and then constructed into pGGDestISceIEG-4sgRNA plasmid, and then the plasmid was co-injected with I-SceI endonuclease and rCutSmart buffer into Tg( zpc :zcas9) zebrafish 1-cell embryos were screened and cultured to obtain heritable F0 generation females, and then trmt61a Zebrafish obtained by hybridization of heterozygous mutant male fish trmt61a Maternal zygotic mutants.

[0058] Tg (zpc: zcas9) transgenic zebrafish were constructed according to the construction method disclosed in the reference:

[0059]

[0060] The Tg ( zpc zcas9) transgenic zebrafish were provided by Shao Ming's laboratory, School of Life Sciences, Shandong University.

[0061] Example 1 Zebrafish trmt61a Determination of spatiotemporal mRNA expression profiles.

[0062] Wild-type zebrafish embryos at different stages, including 2 cell, 8 cell, 1k cell, shield, 24 hours after fertilization (24 hpf), 36 hours after fertilization (36 hpf), 2 days after fertilization (2dpf), and 4 days after fertilization (4 dpf), were collected and fixed, and whole embryo in situ hybridization was used to detect trmt61a The expression of mRNA during zebrafish development; the results showed that trmt61a mRNA is widely expressed from the early embryonic stage (high expression before 1k cells), and gradually decreases with development, e.g. Figure 1 shown.

[0063] Example 2 Synthesis and screening of CRISPR / Cas9 gene knockout target sites.

[0064] 1. Design principles

[0065] The target site should be selected in the first 2 / 3 of the gene coding sequence and after the start codon, and it is best to destroy important domains and / or all transcripts.

[0066] Design 5 targeted zebrafish trmt61a The gene sgRNA sequences are sgRNA0, sgRNA1, sgRNA2, sgRNA3 and sgRNA4. The target sites of these five sgRNA sequences are all located in trmt61a The second exon region of chromosome 13, such as Figure 2 shown.

[0067] The nucleotide sequence of sgRNA0 is sequence 1, and the PAM region connected to the 3' end of sequence 1 is GGG; the nucleotide sequence of sgRNA1 is sequence 2, and the PAM region connected to the 3' end of sequence 1 is GGG; the nucleotide sequence of sgRNA2 is sequence 3, and the PAM region connected to the 3' end of sequence 3 is CGG; the nucleotide sequence of sgRNA3 is sequence 4, and the PAM region connected to the 3' end of sequence 4 is TGG; the nucleotide sequence of sgRNA4 is sequence 5, and the PAM region connected to the 3' end of sequence 5 is TGG;

[0068] 2. Synthesis process.

[0069] 1. Prepare the DNA template required for synthesizing sgRNA.

[0070] The PCR system is as follows:

[0071]

[0072] Among them, the DNA template required for synthesizing sgRNA0, the upstream primer and the downstream primer are T7-trmt61a-F and T7-trmt61a-R respectively, the nucleotide sequence of T7-trmt61a-F is shown in Sequence 24, and the nucleotide sequence of T7-trmt61a-R is shown in Sequence 25.

[0073] The DNA template required for synthesizing sgRNA1, the upstream primer and the downstream primer are T7-trmt61a-F1 and T7-trmt61a-R, respectively, and the nucleotide sequence of T7-trmt61a-F1 is shown in SEQ ID NO: 26;

[0074] The DNA template required for synthesizing sgRNA2, the upstream primer and the downstream primer are T7-trmt61a-F2 and T7-trmt61a-R, respectively, and the nucleotide sequence of T7-trmt61a-F2 is shown in SEQ ID NO: 27;

[0075] The DNA template required for synthesizing sgRNA3, the upstream primer and the downstream primer are T7-trmt61a-F3 and T7-trmt61a-R, respectively, and the nucleotide sequence of T7-trmt61a-F3 is shown in SEQ ID NO:28;

[0076] The DNA template required for synthesizing sgRNA4, the upstream primer and the downstream primer are T7-trmt61a-F4 and T7-trmt61a-R, respectively, and the nucleotide sequence of T7-trmt61a-F4 is shown in SEQ ID NO:29;

[0077] The PCR reaction program is as follows:

[0078]

[0079] The nucleotide sequence of the gRNA scaffold fragment in the pMD19-gRNA scaffold plasmid template used in sgRNA synthesis from the 5' end to the 3' end is shown in Sequence 30;

[0080] 2. In vitro transcription:

[0081] The sgRNA in vitro transcription system is as follows:

[0082]

[0083] The in vitro transcription conditions were: 37°C water bath for 2.5 hours. After the transcription was completed, 1 μL was taken for electrophoresis to detect whether the sgRNA was successfully synthesized.

[0084] 3. Purification of sgRNA.

[0085] Use the mirVana™ miRNA Isolation Kit (Ambion) to recover small RNA fragments: dilute the sgRNA transcription system (combine two 20 μL systems for each sgRNA) to 300 μL with RNase-free water, add 330 μL of anhydrous ethanol; add the solution to the recovery column and centrifuge at 10,000 g for 15 s; add 700 μL of miRNA Wash Solution I and centrifuge for 5-10 s; add 500 μL of Wash Solution II and centrifuge for 5-10 s; repeat once; discard the liquid in the collection tube and centrifuge for 1 min to remove the residual liquid; add an appropriate amount of RNase-free water preheated at 95°C, centrifuge at maximum speed for 20-30 s, and collect the sgRNA solution; take 1 μL of each sgRNA to measure the concentration.

[0086] 4. Microinjection.

[0087] The sgRNA was mixed with Cas9 protein and injected into wild-type or Tg( zpc:zcas9) zebrafish embryos, and some uninjected embryos from the same batch were used as controls, with the final concentrations of sgRNA being 200 ng / μL, the final concentration of Cas9 protein being 200 ng / μL, and the total volume being 1 nL.

[0088] 5. Detect sgRNA efficiency.

[0089] One day after injection, embryos with normal phenotypes were obtained, and genomic DNA was extracted. After PCR amplification, it was sent for sequencing. The primers were trmt61a GT F and trmt61a GT R. trmt61a GT F is shown in sequence 31, and trmt61a GT R is shown in sequence 32;

[0090] The effectiveness and efficiency of sgRNA can be determined based on the peak situation. Figure 3 As shown, after the injection of sgRNA0, the sequencing results indicated that a double peak appeared after the sgRNA0 target sequence, indicating that it was effective; Figure 4 As shown, after the injection of sgRNA1, sgRNA2, sgRNA3 and sgRNA4, the genome of zebrafish embryos with 3 embryos in each tube was used as a template for PCR. Sequencing indicated that a double peak appeared after the sgRNA target site, indicating that the sgRNA was effective in at least 1 of the 3 embryos, indicating that the sgRNA efficiency was high.

[0091] The F0 generation adult fish with wild-type background were crossed with wild-type zebrafish, and the identification primers trmt61a GT F and trmt61a GT R were used for PCR amplification and sequencing identification to obtain the "-4bp" type F1 generation zebrafish; the sequencing sequence specifically includes the following nucleotide sequence as shown in sequence 33, and this nucleotide sequence is named -4bpF1;

[0092] The "-4bp" type adult F1 generation zebrafish females and males were inbred, and the F2 generation was identified and screened by gene sequencing. trmt61a Homozygous mutants and heterozygous mutants, but found trmt61a Homozygous mutants cannot survive to adulthood. trmt61a Heterozygous mutants can survive to adulthood and reproduce; we also found that Western blotting showed that 36 hours after fertilization trmt61a Homozygous mutants have higher levels of Trmt61a protein, such as Figure 5 shown.

[0093] Example 3 Construction of pGGDestISceIEG-4sgRNA plasmid.

[0094] Primers were designed according to the requirements of overlap extension PCR and Gibson assembly, wherein primer fragment 1 F is shown in sequence 6, 1 replacement R is shown in sequence 7, and using pU6sgRNA plasmid as a template, the sgRNA1 sequence was cloned to obtain the U6-sgRNA1 fragment; wherein primer 1 replacement F is shown in sequence 8, fragment 1 R is shown in sequence 9, and using pU6sgRNA plasmid as a template, the sgRNA1 sequence was cloned to obtain the sgRNA1-gRNA scaffold fragment, and then purified; subsequently, using the purified U6-sgRNA1 fragment and the sgRNA1-gRNA scaffold fragment 1:1 mixture as a template, overlap extension PCR was performed using fragment 1 F and fragment 1 R as primers to obtain the U6-sgRNA1-gRNA scaffold fragment, and then purified; the other three sgRNAs were treated in the same manner as above, and primers were designed for sgRNA2, wherein primer fragment 2 F is shown in sequence 10, 2 replacement R is shown in sequence 11, 2 replacement F is shown in sequence 12, and fragment 2 R is shown in sequence 13; primers were designed for sgRNA3, wherein primer fragment 3 F is shown in sequence 14, 3 replacement R is shown in sequence 15, 3 replacement F is shown in sequence 16, and fragment 3 R is shown in sequence 17; primers were designed for sgRNA4, wherein fragment 4 F is shown in sequence 18, 4 replacement R is shown in sequence 19, 4 replacement F is shown in sequence 20, and fragment 4 R is shown in sequence 21.

[0095] Primers were designed according to the requirements of Gibson assembly, wherein vector F was shown in sequence 22, and vector R primer was shown in sequence 23. The pGGDestISceIEG-4sgRNA plasmid was used as a template to amplify the pGGDestISceIEG vector fragment.

[0096] The DNA fragments obtained by PCR were purified using a common DNA product purification kit (TIANGEN) according to the instructions and eluted with 20 μL of double distilled water;

[0097] After obtaining U6-sgRNA1-gRNA scaffold, U6-sgRNA2-gRNA scaffold,

[0098] After the U6-sgRNA3-gRNA scaffold, U6-sgRNA4-gRNA scaffold and pGGDestISceIEG vector fragments were assembled, the concentration was detected using Nanodrop to facilitate the subsequent Gibson assembly.

[0099] The following is the PCR reaction system involved:

[0100] The PCR system for preparing U6-sgRNA1, U6-sgRNA2, U6-sgRNA3 and U6-sgRNA4 fragments is as follows:

[0101]

[0102] The PCR reaction program is as follows:

[0103]

[0104] Preparation of sgRNA1-gRNA scaffold, sgRNA2-gRNA scaffold,

[0105] The PCR reaction system of sgRNA3-gRNA scaffold and sgRNA4-gRNA scaffold fragments is as follows:

[0106]

[0107] The PCR reaction program is as follows:

[0108]

[0109] Preparation of U6-sgRNA1-gRNA scaffold, U6-sgRNA2-gRNA scaffold,

[0110] The PCR reaction system of U6-sgRNA3-gRNA scaffold and U6-sgRNA4-gRNA scaffold fragments is as follows:

[0111]

[0112] The PCR reaction program is as follows:

[0113]

[0114] The PCR reaction system for preparing the pGGDestISceIEG vector fragment is as follows:

[0115]

[0116] The PCR reaction program is as follows:

[0117]

[0118] Example 4 The pGGDestISceIEG-4sgRNA (trmt61a) plasmid was obtained by Gibson assembly.

[0119] After testing the concentration of each fragment, perform Gibson assembly of the five fragments (NEB). According to the instructions, the ratio of the vector fragment to the insert fragment is 1:1, both of which are 0.05 pmol. The required volume of each component is calculated according to the calculation formula recommended in the instructions, and the reaction is carried out according to the following system:

[0120]

[0121] Incubate at 50°C for 60 min, then take 10 μL of the product and transform it into E. coli. After expansion, plate and select single clones to obtain the pGGDestISceIEG-4sgRNA (trmt61a) plasmid. The plasmid map is shown in Figure 6 shown.

[0122] Example 5 Zebrafish trmt61a Preparation of maternal zygotic mutants.

[0123] The preparation process is as follows:

[0124] 1. Use the obtained pGGDestISceIEG-4sgRNA (trmt61a) plasmid, I-SceI endonuclease and rCutSmart buffer to co-inject into Tg( zpc :zcas9) zebrafish 1-cell embryos, where the final concentration of pGGDestISceIEG-4sgRNA (trmt61a) plasmid was 10 ng / μL, the final concentration of I-SceI endonuclease was 1 U / μL, the final concentration of rCutSmart buffer was 0.5×, and the total volume was 1 nL;

[0125] After microinjection, embryos with strong and extensive fluorescence expression were selected under a fluorescence microscope and raised to adulthood to obtain adult zebrafish Tg( zpc : zcas9; U6 :4sgRNA) F0 generation;

[0126] 3. The adult Tg( zpc : zcas9; U6 :4sgRNA) F0 generation was crossed with wild-type zebrafish, and the green fluorescence expression was observed under a fluorescence microscope to screen and obtain transgenic zebrafish Tg( zpc : zcas9;U6 :4sgRNA) F1 generation, such as Figure 7 As shown;

[0127] 4. The transgenic zebrafish Tg( zpc : zcas9; U6 :4sgRNA) F0 generation female fish and existing trmt61a Heterozygous mutant male fish were crossed, and GFP+ embryos were taken to extract genomic DNA. The primers used were trmt61a Mmut iden F and trmt61a GT R, respectively. The nucleotide sequence of trmt61a Mmut iden F from the 5' end to the 3' end is shown in sequence 34. Then, sequencing was performed to determine the type of gene editing, such as Figure 8 As shown;

[0128] 5. trmt61a Whole-embryo in situ hybridization detection of maternal zygotic mutants trmt61a mRNA expression, determined trmt61a The expression disappears completely, e.g. Figure 9 As shown, immunofluorescence detection was performed to confirm that the expression of Trmt61a protein was completely eliminated. Figure 10 shown.

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A zebrafish trmt61a A method for preparing a maternal zygotic mutant, characterized in that: The steps include: S100. Targeting zebrafish trmt61a The sgRNA and Cas9 protein of the gene were co-injected into one-cell embryos of wild-type zebrafish and cultured to adulthood. trmt61a Heterozygous mutants; Among them, the sgRNA is sgRNA0, and its nucleotide sequence is shown in sequence 1; S200. Construct pGGDestISceIEG-4sgRNA plasmid and inject it together with endonuclease and reaction buffer into Tg( zp :zcas9) transgenic zebrafish one-cell embryos were cultured to adulthood to obtain adult zebrafish Tg( zp :zcas9; U6 :4sgRNA) F0 generation; Among them, the pGGDestISceIEG-4sgRNA plasmid includes four sgRNAs, namely sgRNA1, sgRNA2, sgRNA3 and sgRNA4. The nucleotide sequence of sgRNA1 is shown in Sequence 2, the nucleotide sequence of sgRNA2 is shown in Sequence 3, the nucleotide sequence of sgRNA3 is shown in Sequence 4, and the nucleotide sequence of sgRNA4 is shown in Sequence 5; S300. Make adult zebrafish Tg( zp : zcas9; U6 :4sgRNA) F0 generation female fish and trmt61a Heterozygous mutant male fish were crossed, identified, and selected to obtain zebrafish trmt61a Maternal zygotic mutants.

2. The zebrafish as claimed in claim 1 trmt61a A method for preparing a maternal zygotic mutant, characterized in that: In step S100, the preparation process of sgRNA is as follows: S110. Using the pMD19-gRNA scaffold plasmid as a template, using upstream primers and downstream primers, PCR amplification was performed to obtain the DNA template required for synthesizing sgRNA; S120. Generate sgRNA by in vitro transcription using DNA template.

3. The zebrafish as claimed in claim 1 trmt61a A method for preparing a maternal zygotic mutant, characterized in that: The construction of pGGDestISceIEG-4sgRNA plasmid in S200 includes the following steps: S210. Construction of U6-sgRNA1-gRNA scaffold fragment; S220. Construction of U6-sgRNA2-gRNA scaffold fragment; S230. Construction of U6-sgRNA3-gRNA scaffold fragment; S240. Construction of U6-sgRNA4-gRNA scaffold fragment; S250. The constructed U6-sgRNA1-gRNA scaffold fragment, U6-sgRNA2-gRNA scaffold fragment, U6-sgRNA3-gRNA scaffold fragment and U6-sgRNA4-gRNA scaffold fragment were assembled with the pGGDestISceIEG vector fragment to obtain the pGGDestISceIEG-4sgRNA plasmid.

4. The zebrafish as claimed in claim 3 trmt61a A method for preparing a maternal zygotic mutant, characterized in that: The construction of the U6-sgRNA1-gRNA scaffold fragment in S210 includes the following steps: S211. Using pU6sgRNA plasmid as template and sequence 6 and sequence 7 as primers, PCR was used to clone U6 to obtain U6-sgRNA1 fragment; S212. Using pU6sgRNA plasmid as a template and sequence 8 and sequence 9 as primers, the gRNA-scaffold was cloned by PCR to obtain the sgRNA1-gRNA scaffold fragment; S213. Using the mixture of U6-sgRNA1 fragment and sgRNA1-gRNA scaffold fragment as template and sequence 6 and sequence 9 primers, overlap extension PCR was performed to obtain U6-sgRNA1-gRNA scaffold fragment.

5. The zebrafish as claimed in claim 3 trmt61a A method for preparing a maternal zygotic mutant, characterized in that: The construction of the U6-sgRNA2-gRNA scaffold fragment in S220 includes the following steps: S221. Using pU6sgRNA plasmid as template and sequence 10 and sequence 11 as primers, PCR was used to clone U6 to prepare U6-sgRNA2 fragment; S222. Using pU6sgRNA plasmid as template and sequence 12 and sequence 13 as primers, the gRNA scaffold was cloned by PCR to obtain the sgRNA2-gRNA scaffold fragment; S223. Using a mixture of the U6-sgRNA2 fragment and the sgRNA2-gRNA scaffold fragment as a template and sequence 10 and sequence 13 as primers, overlap extension PCR was performed to obtain the U6-sgRNA2-gRNA scaffold fragment.

6. The zebrafish as claimed in claim 3 trmt61a A method for preparing a maternal zygotic mutant, characterized in that: The construction of the U6-sgRNA3-gRNA scaffold fragment in S230 includes the following steps: S231. Using pU6sgRNA plasmid as template and sequence 14 and sequence 15 as primers, PCR was used to clone U6 to prepare U6-sgRNA3 fragment; S232. Using pU6sgRNA plasmid as template and sequence 16 and sequence 17 as primers, the gRNA scaffold was cloned by PCR to obtain the sgRNA3-gRNA scaffold fragment; S233. Using a mixture of the U6-sgRNA3 fragment and the sgRNA3-gRNA scaffold fragment as a template and sequence 14 and sequence 17 as primers, overlap extension PCR was performed to obtain the U6-sgRNA3-gRNA scaffold fragment.

7. The zebrafish according to claim 3 trmt61a A method for preparing a maternal zygotic mutant, characterized in that: The construction of the U6-sgRNA4-gRNA scaffold fragment in S240 includes the following steps: S241. Using pU6sgRNA plasmid as template and sequence 18 and sequence 19 as primers, PCR was used to clone U6 to prepare U6-sgRNA4 fragment; S242. Using pU6sgRNA plasmid as a template and sequence 20 and sequence 21 as primers, the gRNA scaffold was cloned by PCR to obtain the sgRNA4-gRNA scaffold fragment; S243. Using a mixture of the U6-sgRNA4 fragment and the sgRNA4-gRNA scaffold fragment as a template and sequence 18 and sequence 21 as primers, overlap extension PCR was performed to obtain the U6-sgRNA4-gRNA scaffold fragment.

8. The zebrafish according to any one of claims 1 to 7 trmt61a A method for preparing a maternal zygotic mutant, characterized in that: In step S100, the mass ratio of the added amounts of sgRNA and Cas9 is 2:3 to 3:2.

Images