Grass carp-derived U6 promoter and application thereof

Abstract

The invention provides a grass carp sourced U6 promoter. The sequence of the grass carp sourced U6 promoter is shown as one of SEQ ID NO: 1-3. Three grass carp-derived U6 promoters are cloned, the transcriptional activity and cross-species adaptability of the three promoters are analyzed, results show that the three promoters can show transcriptional activity in fish cells and human cells, the transcriptional activity of ciU6-1 is remarkably higher than that of the other two promoters, heterologous DNA can be transcribed in various cells, and the expression of the ciU6-1 in the fish cells and the human cells is improved. And the method can be used for gene editing means based on Cas9.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and particularly relates to a grass carp-derived U6 promoter and application thereof. Background Art

[0002] The CRISPR / Cas system, based on clustered regularly interspaced short palindromic repeats (CRISPR) and its associated proteins (Cas), has ushered in a new era of gene editing technology. The most widely used system is the CRISPR / Cas9 system, which primarily consists of the Cas9 endonuclease and a single guide RNA (sgRNA). By designing specific sgRNAs, this system can cut at the target DNA sequence, creating double-strand breaks, which are then repaired through non-homologous end joining or homologous recombination to achieve genome editing.

[0003] As a revolutionary gene-editing tool, the effective implementation of CRISPR / Cas9 technology relies not only on the precise cutting ability of the Cas9 protein but also, to a large extent, on the efficient expression of sgRNA. In practical applications, specially designed promoters (such as the U6 promoter, H1 promoter, or the T7 promoter for in vitro transcription) are generally used to drive the expression of sgRNA. The mature sgRNA carries a specific targeting sequence, which binds to the Cas9 protein and guides it to precisely recognize the target DNA. Promoter activity influences the expression level of the sgRNA, which in turn affects the efficiency of gene editing.

[0004] The U6 promoter is a typical RNA polymerase III type promoter that can drive the transcription of short RNA molecules with high precision and efficiency, making it of great application value in gene expression regulation and gene editing technology. The U6 promoter has a clear and fixed transcription start site. This feature ensures the uniformity of the transcription product, and is therefore widely used in the expression of small RNAs. As a commonly used driving element, the U6 promoter also plays a key role in CRISPR / Cas9-mediated gene editing. Compared with heterologous promoters from other species, endogenous promoters are more adaptable to the transcriptional environment of the species, thereby effectively reducing the problems of transcriptional regulation differences that may arise when cross-species applications are used. Therefore, in gene editing work, it is particularly important to select and optimize the U6 promoter suitable for the species based on the characteristics of the species.

[0005] Aquaculture is an important part of the agricultural economy. CRISPR / Cas9 gene editing technology has shown broad application prospects in the aquaculture sector, with many applications in the fields of aquatic germplasm improvement, sex determination mechanism regulation, and disease-resistant breeding. The current common practice for gene editing in aquatic animals is to synthesize sgRNA in vitro (using the T7 promoter), assemble it into an RNP complex, and then introduce the RNP complex into fertilized eggs or embryos by microinjection. The introduction of relevant plasmids for gene editing in cultured fish cells by transfection is still not widely used, which limits in-depth research on the functions of important genes in fish. The fish U6 promoter currently in use is mainly derived from the zebrafish promoter. Grass carp is the fish with the largest annual production in my country's freshwater aquaculture, and a gene editing system based on the endogenous U6 promoter of grass carp has not yet been reported. Summary of the Invention

[0006] During our study of the grass carp genome, we cloned three U6 promoters, ciU6-1, ciU6-2, and ciU6-3, and found that all three promoters have transcriptional activity.

[0007] Based on the above research, the present invention provides a U6 promoter derived from grass carp, the sequence of which is shown in one of SEQ ID NOs: 1-3.

[0008] The present invention also provides a gene expression cassette, comprising the above-mentioned U6 promoter and a target gene driven by the U6 promoter.

[0009] In a specific embodiment, the target gene is located downstream of the U6 promoter.

[0010] The present invention also provides an expression plasmid comprising the gene expression cassette for expressing a target gene.

[0011] The present invention also provides the use of the above-mentioned U6 promoter, gene expression cassette or expression plasmid in genetic manipulation of cells.

[0012] In a specific embodiment, the genetic manipulation is gene editing. For example, the U6 promoter can be used for the expression of sgRNA.

[0013] The present invention also provides a method for expressing a target gene in a cell, comprising the step of transferring the above gene expression cassette into the cell.

[0014] In a specific embodiment, the cell is an animal cell.

[0015] In a specific embodiment, the animal cell is a cell of fish origin or a cell of mammalian origin.

[0016] In a specific embodiment, the mammalian-derived cells are human-derived cells.

[0017] The present invention cloned three U6 promoters from grass carp and analyzed their transcriptional activity and cross-species adaptability. The results showed that these three promoters exhibited transcriptional activity in both fish cells and human cells. Among them, the transcriptional activity of ciU6-1 was significantly higher than the other two. It can express heterologous DNA in a variety of cells and can be used for Cas9-based gene editing methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows the sequence alignment of U6 promoters from three grass carp species and a schematic diagram of the conserved motif.

[0019] Figure 2 Schematic diagram of the recombinant plasmid constructed in the embodiment of the present invention. Among them, A is a recombinant plasmid constructed using PX459 as the starting plasmid and driven by U6 promoter sgRNA IRF9 Recombinant plasmid PX459-ciU6-sgRNA IRF9 Schematic diagram; B is the pEGFP-N3 starting plasmid containing sgRNA IRF9 Target sequence fluorescent reporter plasmid pEGFP-sgRNA IRF9 Schematic diagram of the U6 promoter driving sgRNA constructed using PX459 as the starting plasmid. mCherry Recombinant plasmid PX459-ciU6-sgRNA mCherry Schematic diagram of .

[0020] Figure 3 For the recombinant plasmid PX459-ciU6-sgRNA IRF9 with pEGFP-sgRNA IRF9 Fluorescence micrographs of GCO cells after co-transfection.

[0021] Figure 4 For the recombinant plasmid PX459-ciU6-sgRNA IRF9 with pEGFP-sgRNA IRF9 Fluorescence micrographs of co-transfected 293T cells.

[0022] Figure 5 For the recombinant plasmid PX459-ciU6-sgRNA mCherry Transfection of GCF mCherry T7E1 cleavage experiment results (A) and sequencing verification (B) of cells. DETAILED DESCRIPTION

[0023] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0024] 1. Discovery of the Grass Carp U6 Promoter

[0025] The grass carp genome information was analyzed and three U6 genes were obtained. The grass carp cell genomic DNA was extracted, primers were designed, and upstream sequences and possible promoter sequences were amplified to obtain three grass carp U6 promoters, which were named ciU6-1, ciU6-2 and ciU6-3. Their sequences were obtained by sequencing. The sequences are shown in SEQ ID NO: 1-3, respectively. The sequence alignment is shown in Figure 1 As shown, in these promoter sequences, the proximal regions all have a TATA box near the -25 site and a PSE near the -60 site, and the distal regions all contain an OCT element.

[0026] 2. Construction of reporter gene recombinant plasmid for detecting U6 promoter activity

[0027] A reporter gene recombinant plasmid was constructed to detect U6 promoter activity. The fluorescent reporter system used to evaluate U6 promoter efficiency consists of a Cas9 / sgRNA co-expression plasmid (knockout plasmid) and a fluorescent reporter plasmid.

[0028] The Cas9 / sgRNA co-expression plasmid uses the PX459 vector as a backbone, contains a Cas9 expression cassette driven by a CMV promoter, and a puromycin resistance gene, which can be used to enrich cells after transfection. The original human U6 promoter in the plasmid is replaced by the ciU6-1, ciU6-2, and ciU6-3 promoters, respectively named PX459-ciU6-1-sgRNA. IRF9 、PX459-ciU6-2-sgRNA IRF9 、PX459-ciU6-3-sgRNA IRF9 , collectively referred to as PX459-ciU6-sgRNA IRF9 ( Figure 2 A).

[0029] Fluorescent reporter plasmid pEGFP-sgRNA IRF9 Using pEGFP-N3 as a template, a sequence targeting the grass carp IRF9 gene (including the PAM sequence) was inserted into the coding sequence of EGFP, and a 20 bp sequence identical to the sequence before the insertion site was introduced behind the target sequence ( Figure 2 B).

[0030] Due to the introduction of a 43 bp sequence, the EGFP coding sequence was frameshifted. IRF9There will be no green fluorescence. IRF9 After plasmid co-transfection, genome excision and repair mediated by CRISPR / Cas9 system, pEGFP-sgRNA IRF9 By restoring the full green fluorescent protein (GFP) coding sequence through homologous recombination and restoring GFP expression, the effect of gene editing can be detected. Therefore, the transcription efficiency of the U6 promoter can be compared by observing the amount of green fluorescence.

[0031] The specific construction method is as follows: the PX459 vector is linearized using primers PX459-F / R, and the sgRNA sequence of the vector is replaced with the sgRNA sequence targeting the grass carp IRF9 gene (5'-GACGGAGGGACAGGTGACGG-3'). The three U6 promoter fragments are respectively connected with the linearized PX459 vector by homologous recombination to construct the recombinant plasmid PX459-ciU6-1-sgRNA IRF9 、PX459-ciU6-2-sgRNA IRF9 、PX459-ciU6-3-sgRNA IRF9 , this series of plasmids is collectively referred to as PX459-ciU6-sgRNA IRF9 .

[0032] Using pEGFP-N3 as the vector backbone, primers GFP-IRF9-F / R were used for amplification to construct the fluorescent reporter plasmid pEGFP-sgRNA. IRF9 At the same time, the nucleic acid sequence (5'-GACGGAGGGACAGGTGACGGTGG-3') specifically targeting the grass carp IRF9 gene was integrated into the EGFP coding region to make pEGFP-sgRNA IRF9 After being cut, homologous recombination repair is performed, and a 20 bp nucleotide fragment (5'-CGAGGGCGAGGGCGATGCCA-3') that is consistent with the upstream sequence of the insertion site is introduced downstream of the target sequence.

[0033] 3. Detection of Transcriptional Activity of Three U6 Promoters in Fish-Derived Cells

[0034] Each PX459-ciU6-sgRNA IRF9 The recombinant plasmids were combined with pEGFP-sgRNA IRF9 The reporter plasmid was co-transfected into grass carp ovary cells (GCO) at a ratio of 1:1 to transfect only pEGFP-sgRNA. IRF9 cells served as negative control.

[0035] 72 hours after transfection, the green fluorescence expression of cells in each group was observed under a fluorescence microscope. Figure 3 As shown, green fluorescence was observed in cells from all three experimental groups, indicating that all three U6 promoters can effectively drive sgRNA expression. However, there were significant differences in the number of fluorescent cells between the different groups, with the ciU6-1 group being the most significant, with its green fluorescence intensity significantly higher than that of the other groups.

[0036] 4. Detection of Transcriptional Activity of Three U6 Promoters in Human Cells

[0037] Three PX459-ciU6-sgRNA IRF9 Recombinant plasmid and reporter plasmid pEGFP-sgRNA IRF9 Co-transfected into HEK293T cells to transfect only pEGFP-sgRNA IRF9 The cells were used as negative control. After 48 hours of transfection, the green fluorescence expression of cells in each group was observed by fluorescence microscopy. Figure 4 As shown, all experimental groups observed obvious green fluorescence expression in HEK 293T cells, while only pEGFP-sgRNA was transfected IRF9 The negative control group of plasmid showed no green fluorescence.

[0038] The above experimental results show that the three U6 promoters of the present invention can not only drive sgRNA expression in fish cells, but also work in human cells, demonstrating a certain degree of cross-species applicability. When further comparing the number of fluorescent cells in each experimental group, it was found that the trend was basically consistent with the results observed in the GCO cell transfection experiment. The grass carp ciU6-1 promoter also exhibited much higher transcriptional activity in HEK 293T cells than the other two U6 promoters. This shows that the grass carp U6 promoter ciU6-1 can maintain high transcriptional activity in different cell types, which is also of great significance for cross-species gene editing applications.

[0039] 5. Grass carp U6 promoter ciU6-1 edits the mCherry gene integrated into the GCF cell genome

[0040] We have previously established a grass carp fin ray cell line GCF stably transfected with the mCherry gene. mCherry Therefore, we chose to knock out the mCherry gene that had been expressed in the stably transfected cell line to verify the efficiency of the CRISPR / Cas9 system constructed in this article.

[0041] Using mCherry as the target gene, the target site 5'-GTGATGAACTTCGAGGACGG-3' was selected to construct a knockout vector named PX459-ciU6-sgRNA mCherry( Figure 2 C) Using primers mCherry-F / R for template PX459-ciU6-sgRNA IRF9 PCR amplification was performed. The amplified product was recovered to obtain the linearized PX459-ciU6-1 vector, which was then seamlessly cloned and transformed into E. coli Fast-T1 competent cells. Single clones were picked and sequenced for verification.

[0042] Extract the endotoxin-free plasmid and transfect it. 72 hours after transfection, add puromycin to screen the cells. After 5 days, it was observed that all the cells in the control group died, so the screening was terminated, and the transfected cells were collected to extract DNA for T7E1 detection. In order to ensure that the observation of the cut bands during electrophoresis is more intuitive, the mutation site is avoided in the middle of the amplified fragment when designing the primers. This can effectively make the cut product form two bands of different sizes, thereby forming a clearly identifiable separation state in the electrophoresis results. Use specific primers mCherry-f / mCherry-sg4-r to amplify the fragment containing the target (922bp) from the genomic DNA and recover it. The gel-recovered product is then slowly denatured and annealed to form a heteroduplex.

[0043] After the annealing reaction was completed, 1 μL of T7 Endonuclease I was added to the annealing product to perform heteroduplex cleavage. After incubation at 37° C. for 15 min, the cleavage product was directly detected by 2% agarose gel electrophoresis.

[0044] The electrophoresis results after T7E1 digestion showed that PCR successfully amplified a single 922bp target gene band. Compared with the blank control group (NC), the experimental group (sgRNA mCherry ) Two additional bands smaller than 922 bp appeared, approximately 276 bp and 626 bp ( Figure 5 A) demonstrates the effective editing of the mCherry gene by the CRISPR / Cas9 system and verifies that the ciU6-1 promoter can effectively drive the expression of sgRNA, thereby successfully achieving gene editing.

[0045] The PCR amplified product was recovered and ligated to the pMD18-T vector. The E. coli was transformed and single clones were picked for sequencing to verify the mutations caused by gene editing. The sequencing results showed that two types of mutations were generated, one was a base deletion at 642bp in the mCherry gene coding sequence, and the other was a base deletion in the 643-659bp range ( Figure 5 B).

[0046] 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 in the scope of protection of the present invention.

Claims

1. A U6 promoter derived from grass carp, characterized in that: The sequence is shown in one of SEQ ID NOs: 1-3.

2. A gene expression cassette, characterized in that The invention comprises the U6 promoter according to claim 1 and a target gene driven by the U6 promoter.

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

4. An expression plasmid for expressing a target gene, characterized in that: Comprising the gene expression cassette according to claim 2 or 3.

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

6. The use according to claim 5, characterized in that The genetic manipulation is gene editing.

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

8. The use according to claim 5 or 6 or the method according to claim 7, characterized in that The cells are animal cells.

9. The use or method according to claim 8, characterized in that The animal cells are cells derived from fish or mammals.

10. The use or method according to claim 9, characterized in that The mammalian-derived cells are human-derived cells.

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