Production of a zebrafish model of trmt61a enzymatic activity loss

Abstract

This invention relates to the field of gene technology, and more particularly to the preparation of a zebrafish model lacking Trmt61a enzyme activity, for the purpose of preparing a stable genetic model. trmt61a D181A -P2A-EGFP Knock-in zebrafish strains. This invention utilizes CRISPR / Cas9 technology to... trmt61a d181a trmt61a d181a The mutation site was specifically introduced into the zebrafish genome, resulting in the loss of Trmt61a catalytic activity and tRNA m 1 A zebrafish model with significantly reduced tRNA modification levels. This zebrafish model fills a research gap and provides a basis for tRNA m... 1 A provides a usable in vivo research model for studying the biological developmental regulatory mechanisms related to epigenetic modifications and elucidating the pathological mechanisms of diseases (such as malignant tumors and hematopoietic dysfunction), which can be directly used to explore the core role of this epigenetic modification in development and disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic technology, and particularly relates to preparation of a zebrafish model with Trmt61a enzyme activity loss. BACKGROUND

[0002] N1-methyladenosine (m 1 A) is one of the most common modifications on transfer RNA (tRNA). Among them, the m 1 A58 modification is catalyzed by a methyltransferase complex containing two subunits Trmt61a and Trmt6, and Trmt61a is the core subunit that exerts catalytic activity. In in vitro experiments of mammals, it has been confirmed that the aspartic acid at position 181 of Trmt61a is the key catalytic active site.

[0003] tRNA m 1 A modification is mediated by Trmt61a, and its disorder is involved in the regulation of mammalian hematopoietic stem cell stemness maintenance and aging, the regulation of fungal pathogenicity, and the occurrence and development of malignant tumors, so tRNA m 1 A modification is an important regulatory factor in biological development and disease progression.

[0004] Zebrafish is a widely used model animal with high homology to human genes, and the advantages of model organisms are outstanding, which means that the research results usually have important reference value for human related research. By introducing specific mutations related to human diseases into the zebrafish genome, the gene mutations related to human diseases can be simulated, and the functions of these genes in the occurrence and development of diseases can be studied in vivo, the functions of specific gene mutations in organ formation and cell development can be explored, and the foundation for protein function research can be laid. At the same time, it can also provide an ideal animal model for screening and evaluating potential therapeutic drugs.

[0005] However, the current zebrafish model for studying the role of tRNA m 1 A in biological development and disease progression is in a loss state. Therefore, it is urgent to develop a biological development and disease related research model for tRNA m 1 A. SUMMARY

[0006] The present application aims to at least solve one of the technical problems in the related art. To this end, the purpose of the present application is to provide a preparation of a zebrafish model with Trmt61a enzyme activity loss.

[0007] In order to achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows:

[0008] Preparation of a Trmt61a enzyme activity loss zebrafish model for preparing trmt61a D181A -P2A-EGFP Knock-in zebrafish strain.

[0009] Further, the step of:

[0010] S100, using seamless DNA fragment splicing technology, constructing trmt61a D181A -P2A-EGFP- trmt61a- 3'UTR donor plasmid;

[0011] S200, injecting trmt61a D181A -P2A-EGFP- trmt61a 3'UTR donor plasmid, gRNA targeting trmt61a- gene and Cas9 protein into wild type zebrafish embryos at a cell stage, screening and culturing to obtain trmt61a D181A trmt61a Knock-in zebrafish strain.

[0012] Further, in step S100, the seamless DNA fragment splicing technology includes overlap extension PCR and Gibson assembly technology.

[0013] Further, in step S100, -P2A-EGFP D181A -P2A-EGFP- trmt61a Preparation of 3'UTR donor plasmid, including the following steps:

[0014] S110, designing primers according to the requirements of overlap extension PCR and Gibson assembly technology;

[0015] Among them, the primers include Intron 2 F, Intron 2 R, CDS F, Trmt61a D181A R, CDS R, Trmt61a D181A F, 3UTR F, 3UTR R, P2A F, P2A R, GFP F, GFP R, VECTOR F and VECTOR R;

[0016] S120, PCR amplification is carried out by using primers Intron 2 F and Intron 2 R to obtain intron sequence fragment;

[0017] PCR amplification is carried out by using primers CDS F and Trmt61a D181A R to obtain the first half of the Trmt61a CDS sequence fragment carrying D181A point mutation;

[0018] PCR amplification was performed using primers CDS R and Trmt61a D181A F to obtain the latter half of the Trmt61a CDS sequence fragment carrying the D181A point mutation.

[0019] The first half and the second half of the Trmt61a CDS sequence fragment were mixed and then amplified by PCR using primers CDSF and CDSR to obtain the Trmt61a CDS sequence fragment.

[0020] PCR amplification was performed using primers 3UTR F and 3UTR R to obtain Sequence fragments;

[0021] PCR amplification was performed using primers P2A F and P2A R to obtain the P2A sequence fragment;

[0022] PCR amplification was performed using primers GFP F and GFP R to obtain GFP sequence fragments;

[0023] PCR amplification was performed using primers VECTOR F and VECTOR R to obtain the vector sequence fragment;

[0024] S130, Combine the intron sequence fragment and the Trmt61a CDS sequence fragment obtained in step S120. The sequence fragments, P2A sequence fragment, GFP sequence fragment, and vector sequence fragment were assembled using Gibson to obtain... trmt61a- D181A -P2A-EGFP- trmt61a 3'UTR donor plasmid.

[0025] Furthermore, the nucleotide sequence of primer Intron 2 F is shown in SEQ ID NO.1;

[0026] The nucleotide sequence of primer Intron 2 R is shown in SEQ ID NO.2;

[0027] The nucleotide sequence of primer CDS F is shown in SEQ ID NO.3;

[0028] The nucleotide sequence of primer CDSR is shown in SEQ ID NO.4;

[0029] The nucleotide sequence of primer Trmt61a D181A F is shown in SEQ ID NO.5;

[0030] The nucleotide sequence of primer Trmt61a D181A R is shown in SEQ ID NO.6;

[0031] The nucleotide sequence of the primer 3UTR F is shown as SEQ ID NO. 7;

[0032] The nucleotide sequence of the primer 3UTR R is shown as SEQ ID NO. 8;

[0033] The nucleotide sequence of the primer P2A F is shown as SEQ ID NO. 9;

[0034] The nucleotide sequence of the primer P2A R is shown as SEQ ID NO. 10;

[0035] The nucleotide sequence of the primer GFP F is shown as SEQ ID NO. 11;

[0036] The nucleotide sequence of the primer GFP R is shown as SEQ ID NO. 12;

[0037] The nucleotide sequence of the primer VECTOR F is shown as SEQ ID NO. 13;

[0038] The nucleotide sequence of the primer VECTOR R is shown as SEQ ID NO. 14.

[0039] Further, in the mixing process of the first half of the Trmt61a CDS sequence fragment and the second half of the Trmt61a CDS sequence fragment, the two are mixed in equal volume.

[0040] Further, in the step S200, the gRNA targeting trmt61a- The gene is located in the 2nd intron of the gene, and the nucleotide sequence is shown as SEQ ID NO. 15. trmt61a Further, in the step S200, the gRNA targeting

[0041] The gene is prepared by in vitro transcription. trmt61a

[0042] Further, the in vitro transcription comprises the following steps:

[0043] S210, according to the gRNA target site sequence of the gene targeted trmt61a The upstream primer T7-trmt61a-F and the downstream primer gRNA-R are designed.

[0044] The nucleotide sequence of the upstream primer T7-trmt61a-F is shown as SEQ ID NO. 16, and the nucleotide sequence of the downstream primer gRNA-R is shown as SEQ ID NO. 17.

[0045] ​S220. Using pMD19-gRNA scaffold plasmid as a template, PCR amplification was performed using upstream primer T7-trmt61a-F and downstream primer gRNA-R to obtain the double-stranded DNA template required for in vitro transcription of gRNA.

[0046] The nucleotide sequence of the gRNA scaffold fragment in the pMD19-gRNA scaffold plasmid is shown in SEQ ID NO. 18.

[0047] S230 was transcribed and purified in vitro to obtain the targeted... trmt61a gRNA of genes.

[0048] Furthermore, in step S200, the screening and cultivation include the following steps:

[0049] S240. Select embryos with fluorescent hearts under a fluorescence microscope as F0 generation, and culture them until maturity to obtain F0 generation adult fish;

[0050] S250. F0 generation adult fish were crossbred with wild-type zebrafish. Embryos with fluorescent hearts and early trunks were selected for heritability testing to screen for carriers. trmt61a D181A trmt61a Positive embryos were cultured into F1 generation adult fish to obtain -P2A-EGFP D181A trmt61a Knock-in zebrafish strain.

[0051] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0052] This invention provides a method for preparing a zebrafish model lacking Trmt61a enzyme activity, used to establish a stable genetic model. -P2A-EGFP D181A trmt61a Knock-in zebrafish strains. This invention utilizes CRISPR / Cas9 technology to... -P2A-EGFP The mutation site was specifically introduced into the zebrafish genome, resulting in the loss of Trmt61a catalytic activity and tRNA m 1 A zebrafish model with significantly reduced modification levels.

[0053] This zebrafish model fills a gap in existing research and provides a basis for tRNA m 1 A provides a usable in vivo research model for studying the biological developmental regulatory mechanisms related to epigenetic modifications and elucidating the pathological mechanisms of diseases (such as malignant tumors and hematopoietic dysfunction), which can be directly used to explore the core role of this epigenetic modification in development and disease.

[0054] Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following and the accompanying drawings or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0055] trmt61a D181A is provided by the embodiment 1 of the present application Figure 1 D181A The fluorescence picture of F1 generation knock-in zebrafish.

[0056] trmt61a is provided by the embodiment 1 of the present application Figure 2 D181A -P2A-EGFP- trmt61a The schematic diagram of 3'UTR donor plasmid inserting into genome.

[0057] trmt61a- is provided by the embodiment 1 of the present application Figure 3 D181A The display diagram of double side sequencing result of donor plasmid knock-in site in F1 generation zebrafish.

[0058] trmt61a is provided by the verification example 1 of the present application Figure 4 D181A The immunofluorescence picture of F1 generation zebrafish embryo.

[0059] trmt61a is provided by the verification example 2 of the present application Figure 5 D181A / D181A The tRNA m 1 A horizontal column chart. DETAILED DESCRIPTION

[0060] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in connection with specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application. The following embodiments are used to illustrate the present application, but cannot be used to limit the scope of the present application.

[0061] In the following examples, the experimental methods used are conventional methods, and the materials, reagents, etc. used are obtained from commercial channels, unless otherwise specified, according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified.

[0062] The nucleotide sequences involved in the following examples are shown in the following table:

[0063]

[0064] Example 1

[0065] preparation trmt61a D181A trmt61a The process of introducing zebrafish strains is as follows:

[0066] I. Preparation -P2A-EGFP D181A -P2A-EGFP- trmt61a 3'UTR donor plasmid.

[0067] Based on the requirements of overlap extension PCR and Gibson assembly techniques, primers Intron 2 F, Intron 2 R, CDS F, Trmt61a D181A R, CDS R, Trmt61a D181A F, 3UTR F, 3UTR R, P2A F, P2A R, GFP F, GFP R, VECTOR F, and VECTOR R were designed.

[0068] Using the wild-type zebrafish genome as a template, PCR amplification was performed using primers Intron2 F and Intron2 R to obtain the intron 2 sequence fragment.

[0069] Using wild-type zebrafish cDNA as a template, PCR amplification was performed using primers CDSF, Trmt61a D181A R and Trmt61a D181AF, CDSR, to obtain zebrafish carrying the D181A point mutation. trmt61a- The first and second halves of the CDS sequence were extracted, and then these two fragments were mixed in a 1:1 volume ratio. The resulting mixture was used as a template, and PCR was performed using primers CDSF and CDSR via overlap extension to obtain... trmt61a D181A CDS sequence fragments;

[0070] Simultaneously, using wild-type zebrafish cDNA as a template, PCR amplification was performed using primers 3UTR F and 3UTR R to obtain... Sequence fragments;

[0071] Using the P2A-GFF donor plasmid as a template, P2A sequence fragments, GFP sequence fragments, and vector sequence fragments were obtained by PCR amplification using primers P2A F, P2A R and GFP F, GFP R and VECTOR F, and VECTOR R, respectively.

[0072] Through the above steps, partial intron2 sequences were obtained respectively. trmt61a D181AThe nucleotide fragments contained in the CDS sequence, P2A sequence, GFP sequence, 3'UTR sequence, and P2A-GFF vector sequence.

[0073] The intron sequence fragments and Trmt61a CDS sequence fragments obtained above, The sequence fragments, P2A sequence fragment, GFP sequence fragment, and vector sequence fragment were assembled using Gibson sequencing, transformed into E. coli, and single clones were selected to obtain... trmt61a D181A -P2A-EGFP- trmt61a 3'UTR donor plasmid.

[0074] II. Target trmt61a- In vitro transcription of intron 2 gRNA.

[0075] (I) Preparation of DNA templates required for gRNA synthesis.

[0076] The PCR system is shown in the table below:

[0077]

[0078] The nucleotide sequence of the gRNA scaffold fragment in the pMD19-gRNA scaffold plasmid template is shown in SEQ ID NO.18.

[0079] The PCR reaction procedure is shown in the table below:

[0080]

[0081] (II) The in vitro transcription system for gRNA is shown in the table below:

[0082]

[0083] In vitro transcription conditions: 37℃ water bath for 2.5h; after transcription, 1μL of product was taken for electrophoresis to confirm whether gRNA was successfully synthesized. After passing the test, subsequent processing was carried out.

[0084] (III) Purification of gRNA.

[0085] Using mirVana TMThe small fragment RNA was recovered by miRNA isolation kit (Ambion) : the two 20 μL transcription systems of each gRNA were combined, diluted to 300 μL with RNase-free water, 330 μL of anhydrous ethanol was added to obtain a mixed solution; the mixed solution was added to the recovery column, centrifuged at 10,000 g for 15 s; then, miRNA wash solution I (700 μL) was added, centrifuged for 5-10 s; wash solution II (500 μL) was added, centrifuged for 5-10 s, repeated once; the liquid in the kit matched collection tube was discarded, centrifuged for 1 min to remove residual liquid; 30-50 μL of RNase-free water preheated at 95 ℃ was added, centrifuged at the maximum speed for 20-30 s to obtain the in vitro transcribed gRNA.

[0086] III. Culturing of F0 generation adult fish.

[0087] The foregoing constructed trmt61a D181A -P2A-EGFP- trmt61a 3'UTR donor plasmid, synthetic gRNA and Cas9 protein were co-injected into wild type zebrafish embryos at one-cell stage; wherein, trmt61a- D181A -P2A-EGFP- trmt61a The final concentration of 3'UTR donor plasmid was 40 ng / μL, the final concentration of gRNA was 200 ng / μL, and the final concentration of Cas9 protein was 200 ng / μL;

[0088] The embryos with fluorescence in the heart (indicating successful plasmid injection) were selected under a fluorescence microscope as F0 generation and cultured to sexual maturity to obtain F0 generation adult fish.

[0089] The F0 generation adult fish was crossed with wild type zebrafish, and the embryos with green fluorescence in the heart and fluorescence in the early trunk (meeting the trmt61a- transcriptional expression pattern) were selected for heritability detection, and the positive embryos carrying trmt61a D181A trmt61a were screened out and bred into F1 generation adult fish, and the fluorescence map thereof is shown in -P2A-EGFP .

[0090] Genomic DNA was extracted as a template for left arm and right arm PCR, and the positions are shown in Figure 1As shown: left arm PCR was performed using primers Lintron F and Trmt61a D181A R, the nucleotide sequence of primer Lintron F is contained in SEQ ID NO. 19; right arm PCR was performed using primers M13 F and Rintron R, the nucleotide sequences thereof are shown in SEQ ID NO. 20 and SEQ ID NO. 21 respectively. Bands of expected size were amplified in both side PCRs, indicating that PCR amplification was successful; sequencing results of the two side PCR products are shown in Figure 2 As shown, indicating that the insertion is correct.

[0091] Verification Example 1

[0092] F1 generation embryos with green fluorescence expression at about 2.5 hours after fertilization (about 256 cell stage) were taken for immunofluorescence detection, and the results are shown in Figure 3 As can be seen from the figure: GFP is mainly expressed in the nucleus, and has co-localization with the red fluorescently labeled Trmt61a protein, which indicates that the expression of GFP in the knock-in fish line has high specificity;

[0093] In the figure, DAPI is 4', 6-diamidino-2-phenylindole.

[0094] Verification Example 2

[0095] The present application identifies 30 F2 generation Figure 4 D181A / D181A Embryos (F1 generation adult fish output) and controls (wild type zebrafish) were extracted tRNA, and liquid chromatography-mass spectrometry was used to detect m 1 A level, the results are shown in trmt61a Compared with the control group, Figure 5 D181A / D181A Group tRNA m 1 A decreased by about 50%, suggesting that the trmt61a trmt61a D181A Trmt61a enzyme activity of the knock-in fish line is significantly reduced.

[0096] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a zebrafish model lacking Trmt61a enzyme activity, characterized in that, Used for preparation trmt61a D181A -P2A-EGFP The process of knocking in zebrafish strains includes the following steps: S100 utilizes seamless DNA fragment splicing technology to construct... trmt61a D181A -P2A-EGFP- trmt61a- 3'UTR donor plasmid; Seamless DNA fragment splicing technology includes overlap extension PCR and Gibson assembly technology, and includes the following steps: S110. Design primers according to the requirements of overlap extension PCR and Gibson assembly techniques; The primers include Intron 2 F, Intron 2 R, CDS F, Trmt61a D181A R, CDS R, Trmt61a D181A F, 3UTR F, 3UTR R, P2A F, P2A R, EGFP F, EGFP R, VECTOR F, and VECTOR R. The nucleotide sequence of primer Intron 2 F is shown in SEQ ID NO.1; The nucleotide sequence of primer Intron 2 R is shown in SEQ ID NO.2; The nucleotide sequence of primer CDS F is shown in SEQ ID NO.3; The nucleotide sequence of primer CDSR is shown in SEQ ID NO.4; The nucleotide sequence of primer Trmt61a D181A F is shown in SEQ ID NO.5; The nucleotide sequence of primer Trmt61a D181A R is shown in SEQ ID NO.6; S120. PCR amplification was performed using primers Intron 2 F and Intron 2 R to obtain the intron 2 sequence fragment; PCR amplification was performed using primers CDS F and Trmt61a D181A R to obtain the first half of the Trmt61aCDS sequence fragment carrying the D181A point mutation. PCR amplification was performed using primers CDS R and Trmt61a D181A F to obtain the latter half of the Trmt61aCDS sequence fragment carrying the D181A point mutation. The first half of the Trmt61a CDS sequence fragment carrying the D181A point mutation and the second half of the Trmt61a CDS sequence fragment carrying the D181A point mutation were mixed and used as a template. PCR amplification was performed using primers CDSF and CDSR to obtain the Trmt61a D181A CDS sequence fragment. PCR amplification was performed using primers 3UTR F and 3UTR R to obtain Sequence fragments; PCR amplification was performed using primers P2A F and P2A R to obtain the P2A sequence fragment; PCR amplification was performed using primers EGFP F and EGFP R to obtain the EGFP sequence fragment; PCR amplification was performed using primers VECTOR F and VECTOR R to obtain the vector sequence fragment; S130, Take the intron 2 sequence fragment and the Trmt61a D181A CDS sequence fragment obtained in step S120. The sequence fragments, P2A sequence fragment, EGFP sequence fragment, and vector sequence fragment were assembled using Gibson to obtain... trmt61a D181A -P2A-EGFP- trmt61a- 3'UTR donor plasmid; S200, will trmt61a D181A -P2A-EGFP- trmt61a- 3'UTR donor plasmid, targeting trmt61a The gRNA and Cas9 protein of the gene were injected into one-cell-stage wild-type zebrafish embryos, and after selection and culture, the following results were obtained. trmt61a D181A - P2A-EGFP Knock-in zebrafish strains; Among them, targeted trmt61a The gRNA of the gene is located in trmt61a The nucleotide sequence of the second intron of the gene is shown in SEQ ID NO.

15.

2. The method for preparing a zebrafish model lacking Trmt61a enzyme activity as described in claim 1, characterized in that, The nucleotide sequence of primer 3UTR F is shown in SEQ ID NO.7; The nucleotide sequence of primer 3UTR R is shown in SEQ ID NO. 8; The nucleotide sequence of primer P2A F is shown in SEQ ID NO.9; The nucleotide sequence of primer P2A R is shown in SEQ ID NO.10; The nucleotide sequence of primer EGFP F is shown in SEQ ID NO.11; The nucleotide sequence of primer EGFP R is shown in SEQ ID NO.12; The nucleotide sequence of primer VECTOR F is shown in SEQ ID NO.13; The nucleotide sequence of primer VECTOR R is shown in SEQ ID NO.

14.

3. The method for preparing a zebrafish model lacking Trmt61a enzyme activity as described in claim 1, characterized in that, During the mixing process, the first half of the Trmt61a CDS sequence fragment carrying the D181A point mutation and the second half of the Trmt61a CDS sequence fragment carrying the D181A point mutation were mixed in equal volumes.

4. The method for preparing a zebrafish model lacking Trmt61a enzyme activity as described in any one of claims 1 to 3, characterized in that, In step S200, targeting trmt61a The gRNA of a gene is prepared through in vitro transcription.

5. The method for preparing a zebrafish model lacking Trmt61a enzyme activity as described in claim 4, characterized in that, The in vitro transcription includes the following steps: S210, according to the target trmt61a Based on the gRNA target site sequence of the gene, upstream primer T7-trmt61a-F and downstream primer gRNA-R were designed; The nucleotide sequence of the upstream primer T7-trmt61a-F is shown in SEQ ID NO.16, and the nucleotide sequence of the downstream primer gRNA-R is shown in SEQ ID NO.

17. S220. Using pMD19-gRNA scaffold plasmid as a template, PCR amplification was performed using upstream primer T7-trmt61a-F and downstream primer gRNA-R to obtain the double-stranded DNA template required for in vitro transcription of gRNA. The nucleotide sequence of the gRNA scaffold fragment in the pMD19-gRNA scaffold plasmid is shown in SEQ ID NO.

18. S230 was transcribed and purified in vitro to obtain the targeted... trmt61a gRNA of genes.

6. The method for preparing a zebrafish model lacking Trmt61a enzyme activity as described in claim 1, characterized in that, In step S200, the screening and cultivation include the following steps: S240. Select embryos with fluorescent hearts under a fluorescence microscope as F0 generation, and culture them until maturity to obtain F0 generation adult fish; S250. F0 generation adult fish were crossbred with wild-type zebrafish. Embryos with fluorescent hearts and early trunks were selected for heritability testing to screen for carriers. trmt61a D181A -P2A-EGFP Positive embryos were cultured into F1 generation adult fish to obtain trmt61a D181A -P2A-EGFP Knock-in zebrafish strain.

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