Preparation of Trmt61a enzyme activity-deficient zebrafish model

Abstract

The invention relates to the technical field of genes, in particular to preparation of a Trmt61a enzyme activity deleted zebrafish model, and the Trmt61a enzyme activity deleted zebrafish model is used for preparing a zebrafish strain which stably inherits trmt61aD181A-P2A-EGFP knock-in zebrafish strain. According to the invention, trmt61a D181A mutation site specificity is introduced into a zebra fish genome by using a CRISPR / Cas9 technology, and a zebra fish model with Trmt61a catalytic activity deficiency and tRNA m1A modification level significantly reduced is obtained. The zebrafish model fills the existing research blank, provides an available in-vivo research model for biological development regulation mechanism research related to tRNA m1A epigenetic modification and pathological mechanism analysis of diseases (such as malignant tumors and hematopoietic dysfunction), and can be directly used for exploring the core effect of the epigenetic modification in development and diseases.

Description

Technical Field

[0001] This invention relates to the field of gene technology, and in particular to the preparation of a zebrafish model lacking Trmt61a enzyme activity. Background Technology

[0002] N1-methyladenosine monophosphate (m 1 A) is one of the most common modifications on transfer RNA (tRNA). Among them, the m... 1 The A58 modification is catalyzed by a methyltransferase complex containing two subunits, Trmt61a and Trmt6, with Trmt61a being the core subunit responsible for the enzyme's catalytic activity. In vitro experiments in mammals have confirmed that the aspartic acid residue at position 181 of Trmt61a is its key catalytically active site.

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

[0004] Zebrafish are widely used model organisms, sharing high homology with human genes. Their significant advantages as model organisms mean that research findings often have important reference value for human-related studies. By introducing specific mutations associated with human diseases into the zebrafish genome, it is possible to mimic disease-related gene mutations, thereby enabling in vivo studies of the roles of these genes in disease development, exploring the functions of specific gene mutations in organogenesis and cell development, and laying the foundation for protein function research. Furthermore, they provide an ideal animal model for screening and evaluating potential therapeutic drugs.

[0005] However, current research on tRNA m 1 Zebrafish models of the role of tRNA in biological development and disease progression are currently lacking. Therefore, there is an urgent need to develop models targeting tRNA m... 1 A biological development and disease-related research model for A. Summary of the Invention

[0006] The present invention aims to at least solve one of the technical problems existing in related technologies. Therefore, the object of the present invention is to provide a method for preparing a zebrafish model lacking Trmt61a enzyme activity.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Preparation of a zebrafish model lacking Trmt61a enzyme activity, used for preparationtrmt61a D181A -P2A-EGFP Knock-in zebrafish strain.

[0008] Furthermore, it includes the following steps: S100 utilizes seamless DNA fragment splicing technology to construct... 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 wild-type zebrafish embryos at the one-cell stage, and after selection and culture, the following results were obtained. trmt61a D181A -P2A-EGFP Knock-in zebrafish strain.

[0009] Furthermore, in step S100, the seamless DNA fragment splicing technology includes overlap extension PCR and Gibson assembly technology.

[0010] Furthermore, in step S100, trmt61a D181A -P2A-EGFP- trmt61a- The preparation of 3'UTR donor plasmids 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, GFP F, GFP R, VECTOR F, and VECTOR R. S120. PCR amplification was performed using primers Intron 2 F and Intron 2 R to obtain intron sequence fragments; PCR amplification was performed using primers CDS F and Trmt61a D181A R to obtain the first half of the Trmt61a CDS 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 Trmt61a CDS sequence fragment carrying the D181A point mutation. 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. 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 GFP F and GFP R to obtain GFP sequence fragments; PCR amplification was performed using primers VECTOR F and VECTOR R to obtain the vector sequence fragment; 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.

[0011] Furthermore, 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; 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 GFP F is shown in SEQ ID NO.11; The nucleotide sequence of primer GFP 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.

[0012] Furthermore, during 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 volumes.

[0013] Furthermore, in step S200, the target 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.

[0014] Furthermore, in step S200, the target trmt61a The gRNA of a gene is prepared through in vitro transcription.

[0015] Furthermore, 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.

[0016] Furthermore, 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-EGFPPositive embryos were cultured into F1 generation adult fish to obtain trmt61a D181A -P2A-EGFP Knock-in zebrafish strain.

[0017] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: This invention provides a method for preparing a zebrafish model lacking Trmt61a enzyme activity, used to establish a stable genetic model. trmt61a D181A -P2A-EGFP Knock-in zebrafish strains. This invention utilizes CRISPR / Cas9 technology to... 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 modification levels.

[0018] 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.

[0019] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0020] Figure 1 This is provided in Embodiment 1 of the present invention. trmt61a D181A Fluorescent images of F1 knock-in zebrafish.

[0021] Figure 2 This is provided in Embodiment 1 of the present invention. trmt61a D181A -P2A-EGFP- trmt61a- A schematic diagram of the insertion of a 3'UTR donor plasmid into the genome.

[0022] Figure 3 This is provided in Embodiment 1 of the present invention. trmt61a D181A A diagram showing the two-sided sequencing results of the donor plasmid knock-in site in F1 generation zebrafish.

[0023] Figure 4 This is provided by Example 1 of the present invention. trmt61a D181A Immunofluorescence image of F1 generation zebrafish embryos.

[0024] Figure 5 This is provided by verification example 2 of the present invention. trmt61a D181A / D181A zebrafish tRNA m 1 A. Horizontal bar chart. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0026] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0027] The nucleotide sequences involved in the following examples are shown in the table below: Example 1 preparation trmt61a D181A -P2A-EGFP The process of introducing zebrafish strains is as follows: I. Preparation trmt61a D181A -P2A-EGFP- trmt61a- 3'UTR donor plasmid.

[0028] 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. 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. 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. trmt61aThe 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 fragment; Simultaneously, using wild-type zebrafish cDNA as a template, PCR amplification was performed using primers 3UTR F and 3UTR R to obtain... Sequence fragments; 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.

[0029] Through the above steps, partial intron2 sequences were obtained respectively. trmt61a D181A The nucleotide fragments contained in the CDS sequence, P2A sequence, GFP sequence, 3'UTR sequence, and P2A-GFF vector sequence.

[0030] 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.

[0031] II. Target trmt61a In vitro transcription of intron 2 gRNA.

[0032] (I) Preparation of DNA templates required for gRNA synthesis.

[0033] The PCR system is shown in the table below: The nucleotide sequence of the gRNA scaffold fragment in the pMD19-gRNA scaffold plasmid template is shown in SEQ ID NO.18.

[0034] The PCR reaction procedure is shown in the table below: (II) The in vitro transcription system for gRNA is shown in the table below: 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.

[0035] (III) Purification of gRNA.

[0036] Using mirVana TM The miRNA isolation kit (Ambien) was used for small RNA fragment recovery: Two 20 μL transcription systems for each gRNA were combined and diluted to 300 μL with RNase-free water. Anhydrous ethanol (330 μL) was added to obtain a mixed solution. This mixed solution was added to the recovery column and centrifuged at 10,000 g for 15 s. Then, miRNA wash solution I (700 μL) was added and centrifuged for 5–10 s. Wash solution II (500 μL) was added and centrifuged for 5–10 s. This process was repeated once. The liquid in the collection tube provided with the kit was discarded, and centrifuged for 1 min to remove residual liquid. Preheated RNase-free water (30–50 μL) at 95 °C was added and centrifuged at maximum speed for 20–30 s to obtain the in vitro transcribed gRNA.

[0037] III. Cultivation of F0 generation adult fish.

[0038] The aforementioned construction trmt61a D181A -P2A-EGFP- trmt61a- The 3'UTR donor plasmid, synthesized gRNA, and Cas9 protein were co-injected into one-cell-stage wild-type zebrafish embryos; among them, trmt61a D181A -P2A-EGFP- trmt61a- The final concentrations of the 3'UTR donor plasmid were 40 ng / μL, the final concentrations of the gRNA were 200 ng / μL, and the final concentrations of the Cas9 protein were 200 ng / μL. Embryos with fluorescent hearts were selected under a fluorescence microscope (indicating successful plasmid injection) and cultured as F0 generation until sexual maturity to obtain F0 generation adult fish.

[0039] F0 generation adult fish were crossbred with wild-type zebrafish, and embryos with green fluorescence in the heart and early fluorescence in the body (meeting the criteria) were selected. trmt61a Heritability testing was performed on transcriptional expression patterns to screen for carriers. trmt61a D181A -P2A-EGFP Positive embryos were cultured into F1 generation adult fish, and their fluorescence patterns were as follows: Figure 1 As shown.

[0040] Genomic DNA was extracted as a template for PCR in the left and right arms, as shown in the diagram. Figure 2 As shown: PCR on the left arm was performed using primers Lintron F and Trmt61a D181A R, the nucleotide sequence of primer Lintron F is shown in SEQ ID NO. 19; PCR on the right arm was performed using primers M13 F and Rintron R, their nucleotide sequences are shown in SEQ ID NO. 20 and SEQ ID NO. 21, respectively. Both PCRs amplified bands of the expected size, indicating successful PCR amplification; sequencing results of the PCR products from both arms are shown below. Figure 3 As shown, this indicates that the insertion was correct.

[0041] Verification Example 1 Immunofluorescence detection was performed on F1 generation embryos exhibiting green fluorescence expression approximately 2.5 hours post-fertilization (around the 256-cell stage). The results are as follows: Figure 4 As shown in the figure, GFP is mainly expressed in the cell nucleus and co-localizes with the red fluorescently labeled Trmt61a protein. This result indicates that the expression of GFP in the knock-in fish strain is highly specific. In the figure, DAPI is 4',6-diamidinyl-2-phenylindole.

[0042] Verification Example 2 This invention identified 30 F2 generation [items / items]. trmt61a D181A / D181A Embryos (F1 generation adult fish) and controls (wild-type zebrafish) were used to extract tRNA, which was then detected by liquid chromatography-mass spectrometry. 1 Level A, results as follows Figure 5 As shown, compared with the control group, trmt61a D181A / D181A Group tRNA m 1 A decreased by approximately 50%, indicating that the invention constructed... trmt61a D181A The activity of the knock-in fish strain Trmt61a enzyme was significantly reduced.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Preparation of a zebrafish model lacking Trmt61a enzyme activity, characterized in that, Used for preparation trmt61a D181A - P2A-EGFP Knock-in zebrafish strain.

2. The preparation of the zebrafish model lacking Trmt61a enzyme activity as described in claim 1, characterized in that, Includes the following steps: S100 utilizes seamless DNA fragment splicing technology to construct... 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 wild-type zebrafish embryos at the one-cell stage, and after selection and culture, the following results were obtained. trmt61a D181A - P2A-EGFP Knock-in zebrafish strain.

3. The preparation of the zebrafish model lacking Trmt61a enzyme activity as described in claim 2, characterized in that, In step S100, the seamless DNA fragment splicing technology includes overlap extension PCR and Gibson assembly technology.

4. The preparation of the zebrafish model lacking Trmt61a enzyme activity as described in claim 2, characterized in that, In step S100, trmt61a D181A -P2A-EGFP- trmt61a- The preparation of 3'UTR donor plasmids 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, GFP F, GFP R, VECTOR F, and VECTOR R. 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 and the second half of the Trmt61a CDS sequence fragment 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 GFP F and GFP R to obtain GFP sequence fragments; PCR amplification was performed using primers VECTOR F and VECTOR R to obtain the vector sequence fragment; 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.

5. The preparation of the zebrafish model lacking Trmt61a enzyme activity as described in claim 4, characterized in that, 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; 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 GFP F is shown in SEQ ID NO.11; The nucleotide sequence of primer GFP 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.

6. The preparation of the zebrafish model lacking Trmt61a enzyme activity as described in claim 4, characterized in that, During 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 volumes.

7. The preparation of the zebrafish model lacking Trmt61a enzyme activity as described in claim 2, characterized in that, In step S200, targeting 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.

8. The preparation of a zebrafish model lacking Trmt61a enzyme activity as described in any one of claims 2 to 7, characterized in that, In step S200, targeting trmt61a The gRNA of a gene is prepared through in vitro transcription.

9. The preparation of the zebrafish model lacking Trmt61a enzyme activity as described in claim 8, 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.

10. The preparation of the zebrafish model lacking Trmt61a enzyme activity as described in claim 2, 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.

Images