Fish-derived U6 promoter and application thereof

Abstract

The invention provides a micropterus salmoides sourced U6 promoter. The sequence of the promoter is shown as one of SEQ ID NO: 1-4. Four micropterus salmoides-sourced U6 promoters are cloned, the transcriptional activity and cross-species adaptability of the four promoters are analyzed, results show that the four promoters can show better transcriptional activity in fish cells, the transcriptional activity of MsU6-1, 3 and 4 is remarkably higher than that of a human-sourced U6 promoter, and the promoters can be used for exogenous gene expression in the fish cells and can be used for preparing fish cells. The invention further discloses a gene editing means based on Cas9.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a U6 promoter derived from largemouth bass and its applications. Background Technology

[0002] Largemouth bass ( Micropterus salmoides Largemouth bass is a common economic aquaculture species in my country, with low feed costs, high conversion rates, and significant overall economic benefits. However, the farming of largemouth bass is often threatened by some pathogenic microorganisms. Among them, the skin ulcer syndrome caused by largemouth bass iridovirus can lead to a large number of deaths during the farming stage, with a mortality rate as high as 70%.

[0003] Largemouth bass iridovirus ( Largemouth bass ranavirus, LMBV (Large Scale Virus) is a member of the genus Ranavirus in the family Iridoviridae. It is a large, double-stranded DNA virus that widely infects vertebrates. LMBV was initially discovered in wild, natural waters. Currently, LMBV has been successfully isolated from more than 16 fish species, including the European sea bass (Large Scale Virus). Dicentrarchus labrax) ,carp( Cyprinus carpio) Snakehead ( Channa argus These, along with others, are generally susceptible to LMBV and exhibit viral infection and replication. In 2020, Ballard et al. included MCP, ATPase, and DNA polymerase in the core genes of iridoviruses.

[0004] CRISPR / Cas9 gene editing technology is a preferred method for viral control, as it can simply and accurately cut and integrate target gene sequences, and has been widely applied in mammals, fruit flies, and zebrafish. The cleavage action of the CRISPR / Cas9 system can affect the survival of viruses, bacteria, and parasites, and even kill them directly, thus becoming a powerful tool for identifying antiviral targets and developing new therapeutics. The core components of CRISPR / Cas9 technology consist of the sgRNA targeting the gene and the Cas9 nuclease; their efficient delivery and expression are crucial for the use of this technology and the efficiency of gene editing.

[0005] Currently, there are three main delivery forms for sgRNA and Cas9: (1) DNA form, such as DNA plasmids encoding sgRNA and / or Cas9; (2) RNA form, such as in vitro transcribed Cas9 mRNA and sgRNA; and (3) RNP form, such as complexes of sgRNA and Cas9 protein. Among these, using DNA plasmids can achieve durable editing effects, and this technology has been successful in various bony fish, such as zebrafish. Danio rerio ), medaka ( Oryzias latipes ), spotted catfish ( Ictalurus punctatus) and tilapia ( Oreochromis niloticus )wait.

[0006] Stable and efficient promoters are crucial for ensuring the effective expression and function of DNA plasmids after transfection into cells or tissues. The U6 promoter is a typical RNA polymerase III promoter and is widely used in the CRISPR / Cas9 system. U6 promoters in various vertebrates have been characterized, demonstrating significant transcriptional efficiency. In bony fish, U6 promoters have been found in zebrafish and redfin pufferfish (…). Fugu rubripes ), tilapia, and oblique grouper ( Epinephelus coioides The U6 promoter is characterized in the [database name missing], and these promoters also have transcriptional capabilities. Previous studies have shown that species-specific U6 promoters play a crucial role in the expression of the Cas9 protein.

[0007] Currently, the U6 promoter of the largemouth bass has not been identified, limiting the application of CRISPR / Cas9 technology in this species. This invention aims to identify the U6 promoter of the largemouth bass and construct a CRISPR / Cas9 system based on this promoter, laying the foundation for gene function research and genetic improvement in the largemouth bass. Summary of the Invention

[0008] In our study of the largemouth bass genome, we cloned four U6 promoters, all of which exhibited good transcriptional activity.

[0009] Based on the above research, the present invention provides a fish-derived U6 promoter, the sequence of which is shown in one of SEQ ID NO:1-4.

[0010] The present invention also provides a gene expression cassette, comprising the above-mentioned U6 promoter and a target gene driven by the U6 promoter.

[0011] In one specific implementation, the target gene is located downstream of the U6 promoter.

[0012] The present invention also provides an expression plasmid for expressing a target gene, comprising the gene expression cassette.

[0013] This invention also provides the application of the above-mentioned U6 promoter, gene expression cassette, or expression plasmid in genetic manipulation of cells.

[0014] In one specific implementation, the genetic operation is gene editing; for example, the U6 promoter described above can be used for sgRNA expression.

[0015] The present invention also provides a method for expressing a target gene in cells, comprising the step of transferring the above-mentioned gene expression cassette into the cells.

[0016] In one specific implementation, the cell is an animal cell.

[0017] In one specific implementation, the animal cells are fish-derived cells.

[0018] This invention cloned four U6 promoters derived from largemouth bass and analyzed their transcriptional activity and cross-species adaptability. The results showed that these four promoters exhibited good transcriptional activity in fish cells. Among them, MsU6-1, 3 and 4 showed significantly higher transcriptional activity than human U6 promoters. They can be used for exogenous gene expression in fish cells and for Cas9-based gene editing techniques. Attached Figure Description

[0019] Figure 1 Electrophoresis images of PCR amplification products from four U6 promoters derived from largemouth bass (MsU6-1 620bp, MsU6-2 620bp, MsU6-3 620bp, MsU6-4 500bp).

[0020] Figure 2 Activity analysis of four U6 promoters.

[0021] Figure 3 This is a diagram of the recombinant vector MsU6-Px458. Detailed Implementation

[0022] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0023] 1. Cloning and identification of the U6 promoter in largemouth bass By analyzing the genome sequence of the largemouth bass, four potential U6 promoters were screened and identified, including MsU6-1, MsU6-2, MsU6-3, and MsU6-4 (sequences shown in SEQ ID NO: 1-4, respectively). The target fragments were obtained by PCR amplification, and electrophoresis showed that the bands were single and of consistent size (620 bp / 500 bp). Figure 1 The obtained promoter fragments can be used for subsequent activity evaluation and construction of CRISPR-sgRNA drive systems.

[0024] 2. Promoter activity verification The promoter fragments described above were cloned into the pGL4.17 luciferase reporter vector. The constructed luciferase reporter plasmid and the empty pGL4.17 vector were then transfected into EPC using Lipofectamine 3000 liposome transfection reagent. Epithelioma papulosum cypriniIn the cell line (cells), cells were collected and lysed after 24 h, and proteins were extracted for dual-luciferase activity assay. Dual-luciferase assay showed that the reporter gene activity driven by MsU6-1, MsU6-2, MsU6-3, and the truncated MsU6-4 was significantly higher than that of the blank control (different letters indicate significant differences between groups). P <0.05), and the activities of MsU6-1, 3, and 4 were significantly higher than those of the human U6 promoter; among them, MsU6-3 and 4 can be used as the preferred endogenous promoters for subsequent CRISPR-sgRNA systems ( Figure 2 ).

[0025] 3. Modify the CRISPR / Cas9 vector The MsU6-4 promoter of the largemouth bass, which exhibits the best transcriptional activity, was directionally cloned into the px458 plasmid, successfully replacing the original human HU6 promoter, resulting in a full-length 9.5 kb MsU6-px458 recombinant vector (containing SV40 NLS, 3×FLAG, and scaffold sequences). Figure 3 This technology can be used to construct CRISPR / Cas9 gene editing systems for fish such as largemouth bass.

[0026] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A species of largemouth bass ( Micropterus salmoides The U6 promoter from which the source is located is characterized by, The sequence is shown in one of SEQ ID NO:1-4.

2. A gene expression cassette, characterized in that, It includes the U6 promoter as described in claim 1 and the target gene driven by the U6 promoter.

3. The gene expression cassette according to claim 2, characterized in that, The target gene is located downstream of the U6 promoter.

4. An expression plasmid for expressing a target gene, characterized in that, Includes the gene expression cassette as described in claim 2 or 3.

5. The use of the U6 promoter of claim 1, the gene expression cassette of claim 2 or 3, or the expression plasmid of claim 4 in genetic manipulation of cells.

6. The application according to claim 5, characterized in that, The genetic operation in question is gene editing.

7. A method for expressing a target gene in cells, characterized in that, Includes the step of transferring the gene expression cassette of claim 2 or 3 into the cell.

8. The method according to claim 7, characterized in that, The cells in question are animal cells.

9. The method according to claim 8, characterized in that, The animal cells are fish cells.

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