Preparation method of pabpc1a gene deleted zebrafish mutant

Abstract

The invention relates to the technical field of molecular biology, in particular to a preparation method of a pabpc1a gene deleted zebrafish mutant, which is used for preparing the pabpc1a gene deleted zebrafish mutant. According to the preparation method, the CRISPR / Cas9 technology is utilized for the first time to perform targeted knockout on the pabpc1a gene in the zebra fish, and the pabpc1a gene deletion mutant capable of being stably inherited is obtained. The zebrafish mutant with the deletion of the pabpc1a gene, which is constructed and obtained by the invention and can be stably inherited, is expected to be applied to research on the function of the pabpc1a gene, and is expected to be applied to research on an embryonic development molecular mechanism and high-throughput drug screening.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a method for preparing zebrafish mutants with the pabpc1a gene deletion. Background Technology

[0002] In eukaryotic gene expression regulation, polyadenylation of the 3' untranslated region (UTR) of mRNA is a crucial step in ensuring mRNA maturation and function. This modification not only provides the basis for mRNA structural stability but also directly participates in regulating the efficiency of mRNA transport from the nucleus to the cytoplasm. It is also a necessary condition for initiating subsequent translation and a vital bridge connecting post-transcriptional processing and protein synthesis. In this process, members of the polyadenylate binding protein (PABP) family are mainly divided into two categories based on their location and function: nuclear polyadenylate binding proteins (PABPN) located in the nucleus and cytoplasmic polyadenylate binding proteins (PABPC) mainly distributed in the cytoplasm. These two categories play important roles in the nuclear processing and cytoplasmic functional stages of mRNA, respectively. Pabpn precisely regulates the synthesis rate and final length of the polyadenylate (Poly(A)) tail by forming a complex with polyadenylate polymerase (PAP) and other nuclear RNA processing factors. It also assists in the nuclear folding and pre-transport preparation of mature mRNA, ensuring that only mRNAs with a complete Poly(A) tail can be successfully transported into the cytoplasm. Once these polyadenylated mature mRNAs enter the cytoplasm, pabpc rapidly binds to the Poly(A) tail of the mRNA, forming a stable protein-RNA complex, which in turn regulates key functions such as mRNA translation efficiency and stability.

[0003] Although pabpc plays a crucial role in mRNA metabolism, our understanding of its key family subtypes (such as the widely studied pabpc1) remains significantly limited. Existing research on pabpc1 largely focuses on in vitro cell experiments, which struggle to fully simulate the complex tissue microenvironment and signal regulatory networks within organisms. Furthermore, the lack of stable, inherited animal models with pabpc subtype gene deletions directly restricts in-depth research into its physiological functions (such as mRNA regulation during embryonic development and maintenance of tissue homeostasis) and pathological mechanisms (such as functional abnormalities in disease states). In addition, bioinformatics analysis based on cancer genome databases suggests that mutations in some pabpc family subtypes may be potential cancer drivers. These mutations could lead to an imbalance in pabpc's regulatory function on mRNA translation or stability, subsequently causing abnormal expression of downstream target genes and disrupting vital metabolic activities such as cell proliferation and apoptosis, thus providing favorable conditions for tumor growth and metastasis. However, this hypothesis still requires validation using reliable in vivo models. In animal model research, zebrafish embryos are completely transparent, facilitating real-time observation of mRNA expression dynamics and cell development processes. Their small size and high reproduction rate reduce experimental costs and support large-scale studies. Furthermore, the high homology of zebrafish gene sequences with mammalian pabpc family genes ensures that research findings are relevant to human mechanisms. pabpc1a, an important subtype gene in the pabpc family, may be associated with the development and progression of various diseases if its encoded protein is abnormal. However, current research on pabpc1a gene-related diseases still lacks zebrafish models that can accurately simulate disease states. This lack of models hinders research into the pathogenesis of these diseases and the screening of potential therapeutic targets.

[0004] Therefore, constructing an effective research model for pabpc1a based on zebrafish can not only fill the model gap in this field, but also provide important experimental support for subsequent analysis of the in vivo function of the pabpc family, verification of its correlation with diseases, and promotion of targeted drug development and clinical translation. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in related technologies. Therefore, the object of this invention is to provide a method for preparing a zebrafish mutant with the pabpc1a gene deletion.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing zebrafish mutants with pabpc1a gene deletion, used to prepare zebrafish mutants with pabpc1a gene deletion; The pabpc1a gene deletion fragment includes the nucleotide sequence shown in SEQ ID NO.6.

[0007] Furthermore, zebrafish mutants with the pabpc1a gene deletion were prepared using CRISPR / Cas9 technology.

[0008] Furthermore, CRISPR / Cas9 technology includes the following steps: S100. Design gRNA using biological software, the nucleotide sequence of which is shown in SEQ ID NO.1; S200. After mixing gRNA and Cas9 mRNA, the mixture was microinjected into wild-type zebrafish one-cell embryos and cultured to adulthood to obtain the pabpc1a gene knockout zebrafish F0 generation. S300, zebrafish F0 generation with pabpc1a gene knockout were crossbred with wild-type zebrafish, and the resulting embryos were cultured to adulthood to obtain -4bp type F1 generation zebrafish. S400, female and male F1 generation zebrafish of type -4bp were inbred and identified and screened to obtain pabpc1a homozygous mutants. S500. The pabpc1a homozygous mutant was cultured to adulthood to obtain a zebrafish mutant with the pabpc1a gene deleted.

[0009] Furthermore, the gRNA is located on the second exon of the pabpc1a gene.

[0010] Furthermore, during the microinjection process in step S200, the mass ratio of gRNA to Cas9 mRNA injected is 1:3 to 1:4.

[0011] Furthermore, step S200 also includes the following steps for preparing gRNA: S210. Using pMD19-gRNA scaffold plasmid and primer pair I, the DNA template required for gRNA synthesis was obtained by PCR amplification. S220. Using the DNA template, in vitro transcription is performed to obtain crude gRNA; S230. The crude gRNA is purified to obtain pure gRNA.

[0012] Further, in step S210, the primer pair I includes: The upstream primer T7-pabpc1a-F has the nucleotide sequence shown in SEQ ID NO.2; The downstream primer T7-pabpc1a-R has the nucleotide sequence shown in SEQ ID NO.3.

[0013] Furthermore, in step S230, the crude gRNA is purified using a purification kit.

[0014] Furthermore, in step S400, the identification method used is PCR sequencing to verify whether the pabpc1a gene is missing.

[0015] Furthermore, PCR sequencing was performed using primer pair II, which includes: The upstream primer pabpc1a-target3-F has the nucleotide sequence shown in SEQ ID NO.4; The downstream primer pabpc1a-target3-R has the nucleotide sequence shown in SEQ ID NO.5.

[0016] 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 zebrafish mutants with the pabpc1a gene deletion, used to prepare zebrafish mutants with the pabpc1a gene deletion. This method, for the first time, utilizes CRISPR / Cas9 technology to target and knock out the pabpc1a gene in zebrafish, obtaining a stably heritable pabpc1a gene deletion mutant.

[0017] The zebrafish mutant with pabpc1a gene deletion that can be stably inherited by the present invention is expected to be applied not only to the study of pabpc1a gene function, but also to the study of molecular mechanisms of embryonic development and high-throughput drug screening.

[0018] 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

[0019] Figure 1 This is a diagram illustrating the expression of the pabpc1a gene in wild-type zebrafish embryos at different developmental stages, provided in Example 1 of this invention.

[0020] Figure 2 This is a schematic diagram of the gRNA target site sequence and location provided in Embodiment 2 of the present invention.

[0021] Figure 3 This is a diagram showing the electrophoresis results of in vitro transcribed gRNA provided in Example 2 of the present invention.

[0022] Figure 4 This is a sequencing result diagram of wild-type embryos and the pabpc1a homozygous mutant Sanger provided in Example 2 of the present invention.

[0023] Figure 5 This is a diagram showing the mRNA expression of pabpc1a in sibling control embryos and homozygous mutants provided in Example 1 of this invention.

[0024] Figure 6 This is a diagram showing the protein expression of pabpc1a in sibling control embryos and homozygous mutants provided in Example 1 of this invention.

[0025] Figure 7 This is a diagram illustrating the morphological development of zebrafish from sibling control embryos and homozygous mutants provided in Example 2 of this invention. Detailed Implementation

[0026] 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.

[0027] 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.

[0028] The nucleotide sequences involved in the following examples and validation examples are shown in the table below: Example 1 The expression level of pabpc1a in early development of zebrafish was detected as follows: A digoxigenin-labeled antisense RNA probe was synthesized, and pabpc1a expression at different developmental stages of zebrafish embryos was detected using whole embryo in situ hybridization. The results are as follows: Figure 1 As shown in the figure, pabpc1a is expressed from the one-cell stage of the embryo, widely expressed in zebrafish embryos 36 hours after fertilization, and specifically expressed in the hematopoietic tissues of the head and tail 4 days after fertilization. This result suggests that the expression pattern of pabpc1a may play an important role in the early development of zebrafish.

[0029] Example 2 The process for preparing zebrafish mutants with the pabpc1a gene deletion is as follows: I. Design of pabpc1a target (gRNA).

[0030] The design principles are as follows: The Cas9 target site (target) contains 20 bases, with the 5' end being GG (Note: the choice of GG at the 5' end is not a requirement of the Cas9 target itself, but rather due to the T7 promoter used in this experiment for the gRNA in vitro transcription vector. The T7 promoter requires the first two bases of the transcription start site to be GG); the three bases immediately adjacent to the 3' end of the target site constitute the PAM region, requiring the sequence to be NGG (N is any base). The gRNA target was designed on the zebrafish pabpc1a gene using the website https: / / www.benchling.com / crispr. The target should ideally be located in the first two-thirds of the gene coding sequence and after the ATG, but not on the last exon; it should ideally disrupt important domains and / or all transcripts. It should also be considered to avoid the presence of additional start codons with the same reading frame downstream of the first start codon. The target can also be selected at the intron-exon junction to disrupt gene splicing. The 5' UTR and 3' UTR should generally be avoided.

[0031] The designed target site sequences were aligned using the Ensembl website to verify target specificity. Figure 2 As shown in SEQ ID NO.1, the sequence of the zebrafish pabpc1a gene gRNA target site is located in the second exon region of the gene. This figure illustrates the core molecular mechanism in the CRISPR / Cas9 system where gRNA targets and binds to the second exon region of the target gene and uses the PAM sequence to assist Cas9 in DNA recognition and cleavage. This diagram provides a detailed representation of the two strands of the target DNA and its key sequences: Chain of Justice (5'→3' direction): 5'-CCTTCGGGAACATCTTGTCC-3'; Antisense chain (3'→5' direction): 3'-GGAAGCCCTTGTAGAACAGG-5'; gRNA targeting sequence: Through complementary base pairing, it binds to this region of the antisense strand, thereby guiding the Cas9 nuclease to locate the target DNA site; PAM sequence: is a neighboring sequence that the Cas9 enzyme is required to recognize and cut DNA.

[0032] II. Synthesis of gRNA.

[0033] The DNA template required for gRNA synthesis was prepared using the PCR system shown in the table below: The pMD19-gRNA scaffold plasmid template sequence in the table above is shown in SEQ ID NO.7.

[0034] The PCR conditions are shown in the table below: Two 50 μL PCR reaction systems were set up, with a total volume of 100 μL. 1 μL was taken for electrophoresis detection. After the detection was qualified, the obtained PCR product was extracted and purified by gel extraction. Finally, it was eluted with 28 μL of water to obtain the purified DNA template.

[0035] In vitro transcription: The in vitro transcription system for gRNA is shown in the table below. Two 20 μL gRNA in vitro transcription reaction systems were set up, and the transcription conditions were: incubation at 37°C for 150 min. After the transcription reaction, DNase I (1 μL) was added to each reaction system, and the DNA template was digested at 37°C for 40–50 min; then, 0.5–1 μL of the reaction product was taken for electrophoresis to detect the gRNA synthesis. The results are as follows. Figure 3 As shown, this result indicates that the gRNA was successfully synthesized.

[0036] 3. Purify the gRNA.

[0037] Small RNA fragments were recovered using the mirVana™ miRNA Isolation Kit (Ambien): Two 20 μL systems of gRNA products were combined and diluted to 300 μL with RNase-free water. Then, 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 the tube was centrifuged for 1 min to remove residual liquid. RNase-free water preheated to 95 °C (30–50 μL) was added and centrifuged at maximum speed for 20–30 s to obtain the in vitro transcribed gRNA.

[0038] Example 3 The method for constructing zebrafish pabpc1a gene deletion mutants is as follows: I. Microinjection.

[0039] gRNA and Cas9 mRNA were mixed and microinjected into wild-type zebrafish one-cell stage embryos, while some uninjected embryos from the same batch were reserved as sibling controls. The final concentration of gRNA was 150 ng / μL, the final concentration of Cas9 mRNA was 500 ng / μL, and the total volume was 1 μL.

[0040] 2. Detect whether gRNA is effective.

[0041] Five embryos were collected from normal-developing embryos 2-4 days after injection, for a total of 4 groups. Genomic DNA was extracted using the alkaline cleavage method, and the sequence near the gRNA target site was amplified by PCR. Sequencing was then performed, and the peak position was used to determine whether the gRNA was effective.

[0042] The PCR reaction system is shown in the table below: The PCR reaction conditions are shown in the table below: Verification by PCR sequencing confirmed that the prepared zebrafish were F0 generation zebrafish with the pabpc1a gene knocked out.

[0043] III. Preparation of F1 generation zebrafish.

[0044] Adult F0 generation zebrafish were crossed with wild-type zebrafish for heritability and effective mutation detection. F0 crossbred zebrafish capable of producing effective mutations were selected and raised to F1 generation. The caudal fins of these F0 crossbred zebrafish were then clipped (1-2 months). The mutation type was verified by PCR sequencing, resulting in F1 generation zebrafish of the "-4bp" type.

[0045] Adult F1 generation zebrafish of the "-4bp" type were inbred, and homozygous pabpc1a mutants were identified and screened through gene sequencing. The sequencing results are as follows: Figure 4 As shown in the figure, a 4bp deletion is observed at the target site, indicating that the knockout was successful. In the figure, pabpc1a+ / + represents wild-type sibling control, and pabpc1a- / - represents pabpc1a homozygous mutant.

[0046] Test Example 1 Validation of pabpc1a gene expression in homozygous mutants: The expression of pabpc1a mRNA in control embryos and homozygous mutant embryos 28 hours post-fertilization was detected using whole embryo in situ hybridization. The results are as follows: Figure 5 As shown in the figure, pabpc1a expression is lost in the homozygous mutant. Further verification was performed by taking control embryos and homozygous mutant embryos two days after fertilization, respectively, and then performing protein lysis followed by immunoblotting analysis. The results are as follows: Figure 6 As shown in the figure, Pabpc1 protein was not detected in the homozygous mutant, indicating that pabpc1a was completely knocked out in the pabpc1a homozygous mutant. In the figure, Sibling represents sibling control zebrafish, and pabpc1a- / - represents pabpc1a homozygous mutant.

[0047] Test Example 2 Phenotypic analysis of pabpc1a homozygous mutants was performed as follows: Embryo morphology of sibling controls and pabpc1a homozygous mutants was observed at 20 hours and 2 days post-fertilization. Results are as follows: Figure 7 As shown in the figure, the mutant embryos showed blackening (apoptosis) in their head and tail 20 hours after fertilization; 2 hours after fertilization, the mutant embryos had significantly smaller eyes and ears than the control group, and edema in the hindbrain and pericardium, showing obvious developmental delay and deformity. This result indicates that the deletion of the pabpc1a gene leads to abnormal morphological development of zebrafish embryos.

[0048] 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. A method for preparing a zebrafish mutant with pabpc1a gene deletion, characterized in that, Used to prepare zebrafish mutants with pabpc1a gene deletion; The pabpc1a gene deletion fragment includes the nucleotide sequence shown in SEQ ID NO.

6.

2. The method for preparing the pabpc1a gene-deleted zebrafish mutant as described in claim 1, characterized in that, Zebrafish mutants with the pabpc1a gene deletion were prepared using CRISPR / Cas9 technology.

3. The method for preparing the pabpc1a gene-deleted zebrafish mutant as described in claim 2, characterized in that, CRISPR / Cas9 technology includes the following steps: S100. Design gRNA using biological software, the nucleotide sequence of which is shown in SEQ ID NO.1; S200. After mixing gRNA and Cas9 mRNA, the mixture was microinjected into wild-type zebrafish one-cell embryos and cultured to adulthood to obtain the pabpc1a gene knockout zebrafish F0 generation. S300, zebrafish F0 generation with pabpc1a gene knockout were crossbred with wild-type zebrafish, and the resulting embryos were cultured to adulthood to obtain -4bp type F1 generation zebrafish. S400, female and male F1 generation zebrafish of type -4bp were inbred and identified and screened to obtain pabpc1a homozygous mutants. S500. The pabpc1a homozygous mutant was cultured to adulthood to obtain a zebrafish mutant with the pabpc1a gene deleted.

4. The method for preparing the pabpc1a gene-deleted zebrafish mutant as described in claim 3, characterized in that, The gRNA is located in the second exon region of the pabpc1a gene.

5. The method for preparing the pabpc1a gene-deleted zebrafish mutant as described in claim 3, characterized in that, During the microinjection process in step S200, the mass ratio of gRNA to Cas9 mRNA injected is 1:3 to 1:

4.

6. The method for preparing the pabpc1a gene-deleted zebrafish mutant as described in claim 3, characterized in that, Step S200 also includes the following steps for preparing gRNA: S210. Using pMD19-gRNA scaffold plasmid and primer pair I, the DNA template required for gRNA synthesis was obtained by PCR amplification. S220. Using the DNA template, perform in vitro transcription to obtain crude gRNA; S230. The crude gRNA is purified to obtain pure gRNA.

7. The method for preparing the pabpc1a gene-deleted zebrafish mutant as described in claim 6, characterized in that, In step S210, the primer pair I includes: The upstream primer T7-pabpc1a-F has the nucleotide sequence shown in SEQ ID NO.2; The downstream primer T7-pabpc1a-R has the nucleotide sequence shown in SEQ ID NO.

3.

8. The method for preparing the pabpc1a gene-deleted zebrafish mutant as described in claim 6, characterized in that, In step S230, the crude gRNA is purified using a purification kit.

9. The method for preparing the pabpc1a gene-deleted zebrafish mutant as described in claim 3, characterized in that, In step S400, the identification method used is PCR sequencing to verify whether the pabpc1a gene is missing.

10. The method for preparing the pabpc1a gene-deleted zebrafish mutant as described in claim 9, characterized in that, PCR sequencing was performed using primer pair II, which includes: The upstream primer pabpc1a-target3-F has the nucleotide sequence shown in SEQ ID NO.4; The downstream primer pabpc1a-target3-R has the nucleotide sequence shown in SEQ ID NO.5.

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