CN113637670A

CN113637670A - Composition and method for regulating expression of any gene

Info

Publication number: CN113637670A
Application number: CN202110461046.3A
Authority: CN
Inventors: 徐澍; 汪亮; 郭永健
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2021-04-27
Filing date: 2021-04-27
Publication date: 2021-11-12

Abstract

The invention discloses a composition and a method for regulating and controlling expression of any gene. The composition comprises: oligonucleotide probes: the oligonucleotide probe consists of two parts, one part has a nucleic acid secondary structure, the other part is basically complementary with a target substrate, but bases at certain specific positions are not complementary with the target substrate; protein molecule: the protein molecule can recognize the nucleic acid secondary structure of the oligonucleotide probe, so that the protein molecule is combined with the probe and then guided by the probe to be combined with a target substrate to form a ternary compound of target substrate-protein molecule-probe; because the base on a certain specific position of the oligonucleotide probe is not complementary with the target substrate, the protein molecule only has a combination reaction to the target substrate and does not have a cutting reaction. The composition acts on both DNA and RNA targets; can work in prokaryotic cells and eukaryotic cells; can not only up-regulate gene expression quantity, but also down-regulate gene expression quantity.

Description

Composition and method for regulating expression of any gene

Technical Field

The invention belongs to the technical field of molecular biology, and relates to a composition and a method for regulating and controlling expression of any gene.

Background

With the completion of the human genome project, biological research has gradually entered the post-genomic era. Important research topics in the post-genome era are the exploration of gene functions and the search for regulatory networks; whereas systematic characterization of gene function and regulatory networks relies on the precise manipulation of genes by deletion, repression or overexpression. Therefore, DNA and RNA manipulation tools, such as gene editing tools and gene regulatory tools, can deeply advance the development of related research.

Gene editing tools represented by ZFNs, TALENs, CRISPR systems can cut on DNA strands, thereby insertional deletion in the coding region of a target gene, causing a frame shift, which in turn disrupts the function of a specific gene or regulatory element. Recently, we have also reported a novel gene editing tool HpSGN system, which consists of FEN1 protein and a guide probe. Unlike conventional gene editing tools, the HpSGN system has no restriction on the sequence of the target substrate, and the type of target substrate may be either DNA or RNA. However, these "cleavage" methods have drawbacks, and in the case of the HpSGN system, HpSGN destroys the target, resulting in irreversible effects, which cannot be applied to the study of lethal mutations and essential genes. In addition, unnecessary damage may be caused due to cytotoxicity resulting from the formation of a double-strand break in DNA.

Compared with gene editing tools, gene function regulation can be performed in a relatively direct and reversible manner without destroying gene regulation tools that alter target sequences. First, among the existing gene regulation tools, RNAi technology is most widely used to down-regulate the expression level of a target gene by binding to a target RNA, but the technology is limited to application in a specific organism having an appropriate host mechanism. In some organisms, such as prokaryotes, it is not possible to use them. Secondly, another gene regulation tool, CRISPR regulation system CRISPRi/a based on catalytically inactivated Cas protein (dCas), has also been widely used in studies of cell proliferation, drug or toxin sensitivity, and cell transcriptome. Among dCas protein-related families, dCas9 was used to develop CRISPRi system targeting genomic DNA, which can down-regulate gene expression by interfering with the transcription process; and dCasRx is used for developing a target RNA substrate CRISPR system, and can manipulate the change of the expression quantity of genes by interfering the alternative splicing process.

However, to date, there is no gene regulation tool that can simultaneously act on both DNA and RNA targets; can work in prokaryotic cells and eukaryotic cells; can not only up-regulate gene expression quantity, but also down-regulate gene expression quantity. We believe that the feature that FEN1 protein can target DNA and RNA simultaneously can be used to design a gene regulatory system HpSGNi. On the one hand, when the genomic DNA is targeted, the initiation or extension of transcription can be repressed by occupying a promoter region or a coding region, so as to achieve the purpose of down-regulating the gene expression; on the other hand, when targeting miRNA, it is possible to repress the binding to a specific mRNA by occupying miRNA, thereby achieving the purpose of releasing mRNA to up-regulate gene expression. In addition, FEN1 protein has subtype in prokaryotes and eukaryotes, and the species has wide application range.

Disclosure of Invention

The invention aims to provide a novel gene regulation tool and a novel gene regulation method aiming at the defects of the existing gene regulation tool, which can simultaneously act on a DNA target and an RNA target; can work in prokaryotic cells and eukaryotic cells; can not only up-regulate gene expression quantity, but also down-regulate gene expression quantity.

The purpose of the invention can be realized by the following technical scheme:

a composition for the regulation of expression of any gene, comprising two main components:

and (2) component A: oligonucleotide probes: the oligonucleotide probe consists of two parts, one part has a nucleic acid secondary structure; the other part is a single-stranded region that is substantially complementary to the target substrate, but in which bases at certain specific positions are not complementary to the target substrate.

And (B) component: protein molecule: the protein molecule can recognize the nucleic acid secondary structure of the oligonucleotide probe of the component A, so that the protein molecule is combined with the probe and then guided by the probe to be combined with a target substrate to form a ternary compound of target substrate-protein molecule-probe; because the bases at certain specific positions of the oligonucleotide probe are not complementary with the target substrate, the protein molecule only has a binding reaction to the target substrate and does not have a cutting reaction.

The non-complementarity to the target substrate includes both non-complementarity due to the identity with the target substrate and non-complementarity to the target substrate, and in either case, the present invention can be realized as long as the non-complementarity to the target substrate is not present.

In a preferred embodiment of the present invention, the specific position is 1 or more bases of the single-stranded region, preferably 1 to 3 bases of the single-stranded region.

Preferably, the protein molecule comprises a nuclear localization signal or a nuclear export signal.

As a further preferred aspect of the present invention, the composition comprises two main components:

and (2) component A: oligonucleotide probes: the oligonucleotide probe consists of two parts, wherein one part has a stem-loop nucleic acid structure, and the other part is basically complementary with a target substrate, but the base at a certain specific position is not complementary with the target substrate; the number of oligonucleotide probes includes, but is not limited to, 1;

and (B) component: protein molecule: the protein molecule can recognize the stem-loop nucleic acid structure of the oligonucleotide probe, thereby being combined with the probe.

As a further preferred aspect of the present invention, the length of the oligonucleotide probe is not limited, but is preferably 16nt or more.

In a further preferred embodiment of the present invention, the protein molecule is selected from a partial domain or a holoenzyme fragment of one of Afufen, PfuFEN, MjaFEN, MthFEN, and Homo Sapiens FEN.

The composition of the invention is applied to the preparation of gene regulation reagents.

The composition of the invention is applied to gene regulation, and the gene regulation does not include gene regulation aiming at disease diagnosis and/or treatment.

A method for regulating and controlling any gene expression is characterized in that an oligonucleotide probe is designed and synthesized according to a target polynucleotide, the oligonucleotide probe and a protein molecule are added into a system containing the target polynucleotide, the protein molecule recognizes the secondary structure of the oligonucleotide probe, and then is combined with the probe and guided by the probe to be combined with a target substrate to form a 'target substrate-protein molecule-probe' ternary complex; because the base on a certain specific position of the oligonucleotide probe is not complementary with the target substrate, the protein molecule only has a combination reaction to the target substrate and does not have a cutting reaction.

Preferably, the target polynucleotide is DNA or RNA, preferably genomic DNA, plasmid DNA, mRNA or microRNA (also referred to as miRNA).

The beneficial results are that:

first, the novel gene regulation tool and method provided by the invention can act on both a DNA target and an RNA target, and have extremely strong universality.

Secondly, the novel gene regulation tool and the method provided by the invention can work in prokaryotic cells and eukaryotic cells, and have wide applicability.

Thirdly, the novel gene regulation tool and the method provided by the invention can be used for up-regulating the gene expression quantity, can also be used for down-regulating the gene expression quantity, and can also be used for up-regulating and down-regulating different genes.

Fourth, the protein molecules of the invention are smaller than dCas protein in CRISPR regulatory system, which is beneficial to in vivo delivery.

Fifth, the methods of the invention are not limited by the sequence of the target substrate.

Drawings

FIG. 1-schematic diagram of the principle of the present invention and closest prior art

FIG. 2-is a graph showing the results in relation to example 1

FIG. 3-is a graph of the results relating to example 2

FIG. 4-is another graph showing the results in relation to example 2

FIG. 5-is a graph showing the results in relation to example 3

FIG. 6-is a graph showing the results in relation to example 4

FIG. 7-is a graph showing the results in relation to example 5

FIG. 8-is a graph showing the results in relation to example 6

FIG. 9-is a graph showing the results in relation to example 7

FIG. 10-is a graph showing the results in relation to example 8

FIG. 11-is a graph showing the results in relation to example 9

FIG. 12-is a graph showing the results in relation to example 10

FIG. 13-is a graph showing the results in relation to example 11

FIG. 14-is a graph showing the results in relation to example 12

FIG. 15-is a graph showing the results in relation to example 13

Detailed Description

The present invention is described in more detail with reference to the following examples. However, the present invention should not be construed as being limited thereto. The mis-hpDNA probe in the examples is one of the nucleic acid probes described in the present invention. Afufen1 in the examples is one of the protein molecules of the invention.

Example 1 Afufen1 protein is able to bind to, but not cleave, target DNA under the guidance of the mis-hpDNA probe

To verify whether the AffEN 1 protein (nucleotide sequence shown in SEQ ID NO.1 and encoded amino acid sequence shown in SEQ ID NO. 2) could successfully target-bind to the target substrate ssDNA under the guidance of the mis-hpDNA probe, we designed several probes, as shown in FIG. 2, which had a stem-loop structure at the 3 'end and a substantially complementary 5' end to the target ssDNA, but did not match the target ssDNA at certain specific positions. The AffEN 1 protein purified by expression and the probes are respectively incubated for 20min at 37 ℃, then ssDNA is added for incubation for 30min, and the reaction product is subjected to EMSA analysis.

As shown in FIG. 2, when no mismatched bases were present on the probe (PC set, hp4-17), the substrate in the lane was almost completely cleaved (labeled as cleavage product); when the mismatched base on the probe is at position 1(hp4-17-m1), there is no cleavage band in the lane; when the mismatched base on the probe was at position 2(hp4-17-m2), there was a weak cleavage band in the lane; when the mismatched base on the probe was at position 3(hp4-17-m3), there was a more pronounced cleavage band in the lane; when the mismatched bases on the probe were at positions 1 to 3(hp4-17-m), there was no cleavage band in the lane. The above results indicate that Afufen1 is not directed to cleave the target substrate ssDNA when the mismatched bases on the probe are in positions 1 to 3. In addition, as indicated by the dashed box in FIG. 2, although no cleavage reaction occurred, a ternary complex consisting of "ssDNA-probe-FEN 1 protein" was observed, which has a molecular weight of approximately 65kDa (M is protein marker, where the coarsest band is 70 kDa). The above results indicate that Afufen1 can only undergo a binding reaction under the guidance of the probe without cleaving the target substrate ssDNA when the mismatched bases on the probe are at positions 1 to 3. Hereinafter, we refer to this probe with mismatched bases at positions 1 to 3 as mis-hpDNA.

Example 2 Afufen1 protein can efficiently bind to target substrate DNA under the guidance of mis-hpDNA probe

To examine whether AfuFEN1 protein could sufficiently bind to target substrate ssDNA under the guidance of mis-hpDNA, we gradually increased the amount of AfuFEN1 protein, as shown in fig. 3, and the amount of "ssDNA-probe-FEN 1" ternary complex gradually increased with increasing proportion of FEN1 protein to target ssDNA, indicating that AfuFEN1 could better bind to target substrate (ssDNA-S2) under the guidance of mis-hpDNA probe (hp 4-17-m).

To further increase the binding efficiency, we added complexity of the stem-loop structure at the 3' end of the mis-hpDNA probe, version1.0 (hp4-19-m-ver1.0), 1.1(hp4-19-m-ver1.1), 1.2(hp4-19-m-ver1.2) and 1.3(hp4-19-m-ver1.3) probes, respectively. As shown in fig. 4, as the complexity of the stem-loop structure increases, the amount of the "ssDNA-probe-FEN 1" ternary complex also increases gradually. The above results demonstrate that Afufen1 can efficiently bind target substrate DNA under the guidance of the mis-hpDNA probe.

Example 3 Afufen1 protein can bind to target substrate RNA under the guidance of the mis-hpDNA probe

To examine whether AfuFEN1 could bind target substrate RNA under the guidance of the mis-hpDNA probe, we incubated the expression-purified AfuFEN1 protein with mis-hpDNA probes of 1.0(hp4-19-m-ver1.0), 1.1(hp4-19-m-ver1.1), 1.2(hp4-19-m-ver1.2) and 1.3(hp4-19-m-ver1.3) versions at 37 ℃ for 20min, then added target substrate ssRNA (ssRNA-S2) for 30min, and then the reaction product was subjected to EMSA analysis, as shown in FIG. 5. A ternary complex consisting of "ssRNA-probe-FEN 1 protein", marked by the dashed box, was observed; this result demonstrates that AfuFEN1 can efficiently bind to RNA target substrates under the guidance of the mis-hpDNA probe.

Example 4 formation of ternary complexes of ssDNA-Probe-FEN 1 protein with no preference for target substrate sequences

To confirm the preference of the formation of the "ssDNA-probe-FEN 1 protein" ternary complex for the target substrate sequence, we designed probes (hp4-19-1, hp4-19-2, hp4-19-3, hp4-19-4) in different regions of the target substrate (ssDNA-S2) as shown in FIG. 6, and observed whether the generation efficiency of the ternary complex would be different due to the difference in the substrate sequences of the probe binding regions. The experimental results show that the ternary complex of ssDNA-probe-FEN 1 protein can be well formed no matter which region of the target substrate the probe is designed; indicating that the formation of the "ssDNA-probe-FEN 1 protein" ternary complex has no preference for the sequence of the target substrate.

Example 5 Afufen1 protein and mis-hpDNA Probe in prokaryotic cell can regulate the expression of exogenous reporter Gene

If AfuFEN1 protein and the mis-hpDNA probe can regulate and control gene expression in prokaryotic cells, the working mechanism is predicted to be shown in fig. 7-a, on one hand, when the mis-hpDNA probe is combined in the promoter region of a target gene, the combination of RNA polymerase can be inhibited, so that transcription can not be started, and the expression of a target gene is reduced; on the other hand, when the mis-hpDNA probe is combined in the coding region of the target gene, the forward extension of RNA polymerase is inhibited, so that the transcription is not completely completed, thereby down-regulating the expression of the target gene.

In order to verify whether the regulation can be carried out, prokaryotic plasmids expressing Afufen1 protein are transformed into escherichia coli BL21-EGFP cells, and positive clones are screened through resistance; and then the mis-hpDNA probes (EGFP-P1, EGFP-NP1, EGFP-P2, EGFP-NP2, EGFP-T1 and EGFP-T2) which respectively target the promoter region and the coding region of the EGFP gene are electrically transferred into BL21-EGFP-FEN1 cells. And (3) recovering the bacterial liquid after the electrotransformation for 1 hour at the temperature of 37 ℃, adding a tetracycline inducer to induce the expression of FEN1 protein, collecting thalli after inducing the expression for 1 hour, 4 hours and 7 hours respectively, and analyzing the expression quantity of the EGFP by a flow cytometer. As shown in FIG. 7-B, we designed probes at 6 different positions, labeled P1, P2, NP1, NP2, T1, T2. When the mis-hpDNA probe is combined in the promoter region of a target gene (such as P1), the expression of EGFP protein can be observed to be down-regulated by 80%; when the mis-hpDNA probe was bound to the coding region of the target gene (e.g., T1), a 50% down-regulation of EGFP protein expression was observed. The experimental group and the control group have significant difference. The results show that the Afufen1 protein and the mis-hpDNA probe can regulate and control the expression of exogenous reporter genes in prokaryotic cells.

Example 6 Afufen1 protein and mis-hpDNA Probe in prokaryotic cells to control the expression of endogenous genes

In order to verify whether the AffEN 1 protein and the mis-hpDNA probe can regulate and control the expression of endogenous genes in prokaryotic cells, as shown in FIG. 8, a prokaryotic plasmid expressing the AffEN 1 protein is transformed into an Escherichia coli MG1655 cell, and an electrotransformation competent cell is prepared by screening positive clones for resistance; then aiming at the endogenous gene folp and sula promoter regions in Escherichia coli MG1655, designing a mis-hpDNA probe (folp-P1 and sula-P1), electrically transferring the probe into a competent cell, resuscitating the electrically transferred bacterial liquid for 1 hour at 37 ℃, adding a tetracycline inducer to induce the expression of FEN1 protein, collecting the bacterial cells after inducing expression for 4 hours, quantifying the expression levels of folp and sula by using real-time qPCR (primers are folp-F/R, sula-F/R, an internal reference gene is RecA and a primer is RecA-F/R respectively), and the result shows that the expression levels of folp and sula in the experimental group are reduced by about 25 percent compared with the control group. The results show that the Afufen1 protein and the mis-hpDNA probe can regulate and control the expression of endogenous genes in prokaryotic cells.

Example 7 Afufen1 protein and mis-hpDNA Probe inhibit transcription in eukaryotic cells, thereby downregulating the expression of exogenous reporter genes

To verify that AfuFEN1 protein and mis-hpDNA probe can inhibit transcription in eukaryotic cells and thereby down-regulate gene expression, we transfected eukaryotic plasmid expressing AfuFEN1 protein and EGFP plasmid into human kidney cell HEK293A, transfected mis-hpDNA probe targeting respectively to EGFP gene enhancer region and coding region into human kidney cell HEK293A, harvested cells for 48 hours, and analyzed expression amount of EGFP by flow cytometry, as shown in fig. 9. As shown in FIG. 9, it was observed that the expression of EGFP protein was down-regulated by 70-80% regardless of whether the mis-hpDNA probe was bound to the enhancer region of the target gene (e.g., A1, A2, NA1 and NA2) or the mis-hpDNA probe was bound to the coding region of the target gene (e.g., T1, NT1 and T2), and the experimental group was significantly different from the control group. The results show that the Afufen1 protein and the mis-hpDNA probe can regulate and control the expression of exogenous reporter genes in eukaryotic cells.

Example 8 Afufen1 protein and the mis-hpDNA Probe inhibit transcription in eukaryotic cells, thereby downregulating expression of endogenous genes

To verify whether the Afufen1 protein and the mis-hpDNA probe can regulate the expression of endogenous genes in eukaryotic cells, we transfected eukaryotic plasmids expressing Afufen1 protein into human HeLa cells, and then designed four sets of mis-hpDNA probes (CD71-1-version1.0, CD71-1-version1.1, CD71-1-version1.2, CD71-1-version1.3, CXCR4-1-version1.0, CXCR4-1-version1.1, CXCR4-1-version1.2, CXCR4-1-version1.3) for the endogenous genes CD71 and CXCR4 in human genome, as shown in FIG. 10. The expression levels of CD71 and CXCR4 were quantified by real-time qPCR (primers CD71-F/R, CXCR4-F/R, reference gene Gapdh, and primer Gapdh-F/R), and the results showed that the expression levels of CD71 and CXCR4 were down-regulated by about 40% in the experimental group compared with the control group. The results show that the Afufen1 protein and the mis-hpDNA probe can regulate the expression of endogenous genes in eukaryotic cells.

Example 9 Afufen1 protein and mis-hpDNA Probe inhibit translation in eukaryotic cells, thereby downregulating the expression of exogenous reporter genes

To verify that AfuFEN1 protein and mis-hpDNA probe can inhibit translation in eukaryotic cells to down-regulate gene expression, we fused expression nuclear derivation sequence NES at the C-terminal encoding AfuFEN1 protein sequence, constructed eukaryotic plasmid expressing protein only outside the cell nucleus, transfected the constructed plasmid and EGFP plasmid into human kidney cell HEK293A, transfected the mis-hpDNA probe NT1(NT1-version1.2) targeting EGFP gene coding region into human kidney cell HEK293A, harvested cells for 48 hours, and analyzed EGFP expression by flow cytometry, as shown in fig. 11-a. The result is shown in fig. 11-B, the mis-hpDNA probe NT1 targeting the coding region of the EGFP gene can be combined on the mRNA of the EGFP, the experimental group and the control group have significant difference, and the expression of the EGFP protein can be observed to be down-regulated by 62%. As shown in FIG. 11-C, the Afufen1 protein and the mis-hpDNA probe can not only prevent transcriptional elongation in the nucleus by binding to the EGFP genomic coding region, but also prevent translational occurrence outside the nucleus by binding to EGFP mRNA, thereby down-regulating the expression level of EGFP. The above results indicate that AfuFEN1 protein and the mis-hpDNA probe regulate the expression of exogenous reporter genes in eukaryotic cells by inhibiting translation.

Example 10 Afufen1 protein and mis-hpDNA Probe inhibit translation in eukaryotic cells, thereby downregulating expression of endogenous genes

In order to verify whether AffEN 1 protein and a mis-hpDNA probe can inhibit translation in a eukaryotic cell so as to regulate and control the expression of an endogenous gene, as shown in figure 12, a nuclear export sequence NES is fused and expressed at the C end of a protein sequence coding AffEN 1, a eukaryotic plasmid for expressing the protein only outside a cell nucleus is constructed, the eukaryotic plasmid is transfected into a human HeLa cell, and the mis-hpDNA probe (CXCR4-5-version1.3) is designed aiming at mRNA of the endogenous gene CXCR4 in a human genome. The protein level of the CD71 expression level is quantified by a flow cytometer, and the transcription level of the CD71 expression level is quantified by real-time qPCR (the primers are CXCR4-F/R, the internal reference gene is Gapdh and the primers are Gapdh-F/R), so that the results show that the protein expression level of CXCR4 in an experimental group is reduced by about 50% compared with a control group, but the transcription level has no obvious difference change. The results show that the Afufen1 protein and the mis-hpDNA probe can reduce the expression of endogenous genes in eukaryotic cells by inhibiting translation.

Example 11 & Afufen1 protein and mis-hpDNA probe can up-regulate the expression of endogenous gene in eukaryotic cells & all the above examples are down-regulated gene expression level, and in order to examine whether Afufen1 protein and mis-hpDNA probe can up-regulate the expression of endogenous gene in eukaryotic cells, we selected microRNA (miRNA for short) as a target. As shown in fig. 13-a, in eukaryotic cells, certain mirnas can specifically bind to corresponding mrnas to form double-stranded RNA molecules, which in turn cause degradation or inhibit their translation; the Afufen1 protein and the mis-hpDNA probe can be combined with miRNA molecules to form a ternary complex, so that the inhibition effect of miRNA on mRNA is reduced, and the expression quantity of corresponding genes is increased. As shown in FIG. 13-B, studies have shown that the expression of EGFR protein, an endogenous gene, is inhibited by miRNA-21, and we designed a mis-hpDNA probe targeting miRNA-21 (mis-hp-mi21-5p-version1.0, mis-hp-mi21-5p-version1.1, mis-hp-mi21-5p-version1.2, and mis-hp-mi21-5p-version1.3), so as to up-regulate the expression level of EGFR gene by inhibiting miRNA-21. As shown in FIG. 13-B, when Afufen1 protein and the mis-hpDNA probe were transferred into cells, the expression level of EGFR was up-regulated by about 3-fold, which was significantly different from that of the control group. The above results indicate that AfuFEN1 protein and the mis-hpDNA probe can up-regulate the expression of endogenous genes in eukaryotic cells.

Example 12 Afufen1 protein and mis-hpDNA Probe simultaneously Up-and Down-regulated expression of endogenous genes in eukaryotic cells

In order to investigate whether the Afufen1 protein and the mis-hpDNA probe can simultaneously up-regulate and down-regulate the expression of endogenous genes in eukaryotic cells, we designed a mis-hpDNA probe targeting CXCR4 gene mRNA and miRNA-21 (CXCR4-5-version1.3, mis-hp-mi21-5p-version1.3), respectively, as shown in FIG. 14-A; the former can down-regulate the expression of CXCR4 gene by inhibiting transcription, and the latter can up-regulate the expression of EGFR by inhibiting the binding of miRNA-21 to EGFR gene mRNA. The experimental results are shown in fig. 14-B, C and D, when AfuFEN1 protein and the mis-hpDNA probe were transferred into cells, the expression level of CXCR4 was down-regulated by 45%, the expression level of EGFR was up-regulated by about 197%, and there was a significant difference from the control group. The results show that the Afufen1 protein and the mis-hpDNA probe can simultaneously up-regulate and down-regulate the expression of different endogenous genes in eukaryotic cells.

Example 13 comparison of HpSGNi with existing Gene regulatory methods RNAi, ORFs and CRISPII

In some cases, to fully characterize gene function and regulatory networks, we need to simultaneously up-regulate and down-regulate the expression of different genes. For conventional methods, it is necessary to work with a gene up-regulation tool and a gene down-regulation tool in the same cell at the same time, which may cause inconvenience in operation and may even cause interference between different tools. However, there is no gene regulation tool that can up-regulate or down-regulate the expression level of a gene. The advantage of the HpSGNi system over traditional gene regulation methods is that, on the one hand, when targeting genomic DNA, one can repress transcription initiation or extension by occupying the promoter region or coding region, achieving the goal of down-regulating gene expression; on the other hand, when targeting miRNA, it is possible to repress the binding to a specific mRNA by occupying miRNA, thereby achieving the purpose of releasing mRNA to up-regulate gene expression.

To better demonstrate the superiority of the HpSGNi system, we compared the possibility of RNAi, Open Reading Frames (ORFs), CRISPRi and HpSGNi simultaneously up-regulating the expression of EGFR protein and down-regulating the expression of EGFP. In this parallel experiment, HEK293A cells were first transfected with a plasmid encoding EGFP. siRNAs (SiRNA-sense and SiRNA-antisense) targeting the coding region of the EGFP gene, plasmids in which the sgRNAs targeting the coding region of the EGFP gene express dCas9 protein, plasmids targeting the EGFR protein, plasmids expressing FEN1 protein, and probes (mis-hp-mi21-5p-version1.3 and Egfp-NT1-version1.2) targeting the coding region of the EGFP gene and miRNA-21-5p are transfected into each group. Flow cytometry detects expression levels of EGFP and EGFR. As shown in fig. 15, the expression levels of EGFP were down-regulated by about 48%, 0%, 64% and 30% for the siRNA, ORFs, CRISPRi and HpSGNi groups, respectively. Meanwhile, the expression levels of EGFR in the siRNA, ORFs, CRISPR and HpSGNi groups were up-regulated by about 3%, 420%, 0% and 30%, respectively. The HpSGNi system is the only system that can inhibit both EGFP expression and EGFR activity expression.

Sequence listing

<110> university of Chinese pharmacy

<120> a composition and method for arbitrary gene expression regulation

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1011

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgggtgcgg atattggtga cctctttgag agggaagagg tcgagcttga gtacttctca 60

ggaaagaaaa ttgccgttga tgctttcaac acgctatacc agttcatctc gataataagg 120

cagcctgacg gtacgccgtt aaaggactca cagggcagaa tcacctctca cctttccgga 180

atcctataca gagtctccaa catggtcgag gtgggaatca ggccggtgtt tgtattcgac 240

ggagagccac cggagttcaa gaaggctgaa attgaggaga ggaaaaagag aagggctgag 300

gcagaggaga tgtggattgc ggctttgcag gcaggagata aggacgcgaa aaagtatgct 360

caggctgcag ggagggttga cgagtacatt gttgactccg caaagacgct tttaagttac 420

atggggattc cctttgtcga tgccccgtct gaaggagagg cgcaggctgc ttacatggca 480

gcaaaaggcg atgtggagta cacaggaagc caggattacg attctctgct cttcggaagc 540

ccgagactcg ccagaaatct cgcaataacg ggaaaaagga agcttcccgg caaaaatgtc 600

tatgtggatg taaagccgga gataataatt ctggaaagca acctcaaaag gctgggtttg 660

acgagggagc agctcatcga catagcgatt ctggtcggga cggactacaa tgagggtgtg 720

aagggtgtcg gcgtcaagaa ggctttgaac tacatcaaga cctacggaga tattttcagg 780

gcactcaagg ctctgaaagt aaatattgac cacgtagagg agataaggaa tttcttcctg 840

aatcctcctg tgactgacga ctacagaata gagttcaggg agcctgactt tgagaaggcc 900

atcgagttcc tgtgcgagga gcacgacttc agcagggaga gggtcgagaa ggccttggag 960

aagctcaaag ctctgaagtc aacccaggcc acgcttgaga ggtggttctg a 1011

<210> 2

<211> 336

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Gly Ala Asp Ile Gly Asp Leu Phe Glu Arg Glu Glu Val Glu Leu

1 5 10 15

Glu Tyr Phe Ser Gly Lys Lys Ile Ala Val Asp Ala Phe Asn Thr Leu

20 25 30

Tyr Gln Phe Ile Ser Ile Ile Arg Gln Pro Asp Gly Thr Pro Leu Lys

35 40 45

Asp Ser Gln Gly Arg Ile Thr Ser His Leu Ser Gly Ile Leu Tyr Arg

50 55 60

Val Ser Asn Met Val Glu Val Gly Ile Arg Pro Val Phe Val Phe Asp

65 70 75 80

Gly Glu Pro Pro Glu Phe Lys Lys Ala Glu Ile Glu Glu Arg Lys Lys

85 90 95

Arg Arg Ala Glu Ala Glu Glu Met Trp Ile Ala Ala Leu Gln Ala Gly

100 105 110

Asp Lys Asp Ala Lys Lys Tyr Ala Gln Ala Ala Gly Arg Val Asp Glu

115 120 125

Tyr Ile Val Asp Ser Ala Lys Thr Leu Leu Ser Tyr Met Gly Ile Pro

130 135 140

Phe Val Asp Ala Pro Ser Glu Gly Glu Ala Gln Ala Ala Tyr Met Ala

145 150 155 160

Ala Lys Gly Asp Val Glu Tyr Thr Gly Ser Gln Asp Tyr Asp Ser Leu

165 170 175

Leu Phe Gly Ser Pro Arg Leu Ala Arg Asn Leu Ala Ile Thr Gly Lys

180 185 190

Arg Lys Leu Pro Gly Lys Asn Val Tyr Val Asp Val Lys Pro Glu Ile

195 200 205

Ile Ile Leu Glu Ser Asn Leu Lys Arg Leu Gly Leu Thr Arg Glu Gln

210 215 220

Leu Ile Asp Ile Ala Ile Leu Val Gly Thr Asp Tyr Asn Glu Gly Val

225 230 235 240

Lys Gly Val Gly Val Lys Lys Ala Leu Asn Tyr Ile Lys Thr Tyr Gly

245 250 255

Asp Ile Phe Arg Ala Leu Lys Ala Leu Lys Val Asn Ile Asp His Val

260 265 270

Glu Glu Ile Arg Asn Phe Phe Leu Asn Pro Pro Val Thr Asp Asp Tyr

275 280 285

Arg Ile Glu Phe Arg Glu Pro Asp Phe Glu Lys Ala Ile Glu Phe Leu

290 295 300

Cys Glu Glu His Asp Phe Ser Arg Glu Arg Val Glu Lys Ala Leu Glu

305 310 315 320

Lys Leu Lys Ala Leu Lys Ser Thr Gln Ala Thr Leu Glu Arg Trp Phe

325 330 335

Claims

1. A composition for the regulation of expression of any gene, characterized by comprising two main components:

2. The composition of claim 1, wherein the specific position is 1 or more bases of the single-stranded region, preferably 1 to 3 bases of the single-stranded region.

3. The composition of claim 1, wherein the protein molecule comprises a nuclear localization signal or a nuclear export signal.

4. The composition according to claim 2 or 3, characterized by comprising two main components:

and (2) component A: oligonucleotide probes: the oligonucleotide probe consists of two parts, one part has a stem-loop nucleic acid structure, the other part is basically complementary with a target substrate, but bases at certain specific positions are not complementary with the target substrate; the number of oligonucleotide probes includes, but is not limited to, 1;

5. The composition according to claim 4, characterized in that the length of the oligonucleotide probe is not limited, preferably 16nt or more.

6. The composition according to claim 4, wherein the protein molecule is selected from the group consisting of partial domains and holoenzyme fragments of Afufen, PfuFEN, MjaFEN, MthFEN, and Homo Sapiens FEN.

7. Use of the composition of any one of claims 1-3 for the preparation of a gene regulatory agent.

8. Use of the composition of any one of claims 1-3 for gene regulation, excluding gene regulation for disease diagnosis and or treatment.

9. A method for the regulation of expression of any gene, characterized in that an oligonucleotide probe according to any one of claims 1 to 3 is designed and synthesized on the basis of a target polynucleotide, said oligonucleotide probe and a protein molecule according to any one of claims 1 to 3 are added to a system containing the target polynucleotide, said protein molecule recognizes the secondary structure of the oligonucleotide probe, thereby binding to the probe and then being guided by the probe to bind to a target substrate to form a "target substrate-protein molecule-probe" ternary complex; because the base on a certain specific position of the oligonucleotide probe is not complementary with the target substrate, the protein molecule only has a combination reaction to the target substrate and does not have a cutting reaction.

10. The method of editing any target polynucleotide according to claim 9, wherein the target polynucleotide is DNA or RNA, preferably genomic DNA, plasmid DNA, mRNA or microRNA.