Target gene expression control system comprising dxcas9 and CRP derivative and preparation method therefor

Abstract

To provide a plasmid comprising dxCas9 and a CRP derivative for controlling the expression of a target gene.SOLUTION: The present invention relates to a plasmid comprising dxCas9 and a CRP derivative for controlling the expression of a target gene, a recombinant strain transformed with the plasmid, a preparation method therefor, a target gene control system, and a method for controlling the expression of a target gene, wherein the plasmid, recombinant strain, and expression control system according to the present invention can improve the production of high-value-added substances by construction of a system for simultaneously and multiply controlling the expression of target genes.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a plasmid for regulating target gene expression comprising dxCas9 and a CRP derivative, a recombinant strain transformed with the plasmid, a method for producing the same, a target gene regulation system, and a method for regulating target gene expression. [Background technology]

[0002] Currently, in the bioindustry, techniques are widely used to metabolically engineer industrial microorganisms to efficiently produce products that cannot be produced by wild-type microorganisms in order to produce high-value-added products. The core of these techniques is to produce target substances efficiently and economically. The metabolic engineering methods used for this include techniques for inserting heterologous genes onto plasmids or genomes and techniques for mutating specific genes. In particular, in recent years, techniques using CRISPR to rapidly insert or mutate target genes have been attracting attention.

[0003] Since CRISPR technology has the potential to simultaneously and multiplex target endogenous or foreign genes in E. coli, the development of a CRISPR-based gene expression regulation system has great potential as a synthetic biology tool that can improve the production of high-value-added substances by E. coli. However, although there are cases in which CRISPR-based gene expression regulation tools have been developed in eukaryotes to date, there are relatively few reports on a regulation system that simultaneously activates and suppresses gene expression in E. coli.

[0004] Against this background, the present inventors developed a new CRISPR-based gene expression regulation system in Escherichia coli that simultaneously targets multiple genes and has the function of transcriptional activation / repression, thereby completing the present invention. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Pearson et al (1988)[Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-licensed document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-licensed document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0006] The present invention aims to provide a plasmid for regulating target gene expression, which comprises dxCas9 and a CRP derivative.

[0007] Another object of the present invention is to provide a recombinant strain transformed with the plasmid.

[0008] Furthermore, the present invention aims to provide a system for regulating target gene expression, comprising dxCas9 and a CRP derivative.

[0009] Furthermore, the present invention aims to provide a method for producing a recombinant strain that regulates target gene expression, comprising the steps of: i) preparing a dxCas9-CRP system in which a dxCas9 protein and a CRP protein are combined; ii) cloning a fluorescent reporter plasmid into the dxCas9-CRP system prepared as described above; iii) further cloning a guide RNA into the dxCas9-CRP system prepared in step ii); and iv) transforming the dxCas9-CRP-gRNA into a strain.

[0010] Furthermore, the present invention aims to provide a method for regulating target gene expression, which is characterized by including a step of applying a CRP derivative to dxCas9.

[0011] Furthermore, the present invention aims to provide an application of a plasmid containing dxCas9 and a CRP derivative for regulating expression of a target gene.

[0012] Furthermore, the present invention aims to provide an application of a system comprising dxCas9 and a CRP derivative for regulating target gene expression. [Means for solving the problem]

[0013] These will be described in detail below. Note that each description and embodiment disclosed in the present invention is also applicable to other descriptions and embodiments. In other words, all combinations of various elements disclosed in the present invention are included in the present invention. In addition, the present invention is not limited to the following specific description.

[0014] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the present invention.

[0015] One aspect of the present invention provides a plasmid for regulating target gene expression, comprising dxCas9 and a CRP derivative. Effect of the Invention

[0016] The plasmid, recombinant strain and expression regulation system according to the present invention can improve the production of high-added-value substances by constructing a system for simultaneously and multiplexly regulating the expression of target genes. [Brief description of the drawings]

[0017] [Figure 1] This is a design diagram of the plasmid for the CRISPR-based gene expression regulation system and the GFP fluorescent expression plasmid for system performance verification. [Diagram 2] FIG. 1 is a schematic diagram showing the process of enhancing / repressing target gene expression regulation by the CRISPR-based gene expression regulation system. [Diagram 3] FIG. 1 shows the plasmid map of pMW7-PJ23117-GFP. [Figure 4] FIG. 1 shows the plasmid map of pMW7-PJ23119-GFP. [Diagram 5]FIG. 1 shows the plasmid map of pMW7-PJ23117-GFP-PJ23119-mCherry. [Figure 6] FIG. 1 shows the gRNA target position on the fluorescent plasmid (target gene). [Figure 7] FIG. 1 is a cloning blueprint for gRNA expression. [Figure 8] FIG. 1 shows the plasmid map of pdxCas9-CRPAR123-gRNA(A). [Figure 9] FIG. 1 shows the plasmid map of pdxCas9-CRPAR123-gRNA(R1). [Figure 10] FIG. 1 shows the plasmid map of pdxCas9-CRPAR123-gRNA(A / R2). [Figure 11] 1 is a graph comparing the target gene transcription activity efficiency of dxCas9-conjugated CRP derivatives. [Figure 12] 1 is a graph comparing the efficiency of dxCas9-conjugated CRP derivatives in suppressing target gene transcription. [Figure 13] This is a schematic diagram of dxCas9-CRPAR123's enhanced / inhibited regulation of simultaneous GFP / mCherry gene expression, and a graph showing the results of verifying the multiple targeting potential of the dxCas9-CRPAR123 system. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In the present invention, "dCas9 (dead Cas9)" refers to a protein that is capable of sequence-specific recognition / binding, but has inactivated nucleolytic activity, and thus can regulate gene expression without cleaving the target DNA. The dCas9 can regulate target gene expression by recognizing / binding to the PAM sequence consisting of the NGG sequence in the target gene sequence.

[0019] In the present invention, "dxCas9" refers to an evolved mutant Cas9 that has been mutated to increase the specific recognition efficiency in a wide range of PAM sequences (NG, NNG, GAA, GAT, CAA) because dCas9 has the limitation that an NGG sequence must be present at the end of the target site in order to select a target position on a target gene sequence, and thus refers to a protein with improved PAM compatibility.

[0020] In the present invention, dxCas9 includes, but is not limited to, the amino acid sequence represented by SEQ ID NO:1.

[0021] In the present invention, the dxCas9 is linked to a linker comprising the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0022] In the present invention, the dxCas9 and CRP or a CRP derivative may be linked to each other via a linker.

[0023] The base sequence encoding the linker must be of sufficient length. Specifically, the base sequence encoding the linker of the present invention has a length of 20 bp or more, for example, a length of 30 or 40 bp or more. The base sequence encoding the linker has at least about 80% homology with the linear vector, and may have 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0024] In the present invention, the linker includes, but is not limited to, the amino acid sequence GGGAGGGGAG (SEQ ID NO: 2) which is composed of small and simple amino acids of glycine and alanine so that the binding protein has structural flexibility.

[0025] In the present invention, "CRP (cAMP Receptor Protein)" refers to a cAMP receptor protein, which is a transcriptional regulatory protein that promotes or suppresses the transcription of about 100 or more genes in bacteria. DNA binding of CRP brings about RNA polymerase and promotes the transcriptional activity of target genes. CRP is structurally composed of AR1, AR2, and AR3 (activating region) that bind to the α-subunit of RNA polymerase to activate gene transcription. CRP is also called catabolite gene activator protein (CAP). In the present invention, CRP is used as an effect domain of dxCas9, but is not limited thereto.

[0026] In the present invention, the term "derivative" refers to a similar protein derived from a wild-type protein by removing, adding, and / or substituting a specific domain. In particular, the protein may have improved in vivo stability, storage stability, or activity due to the removal, addition, and / or substitution of the amino acid or chemical residue, and may have improved or newly added advantageous properties as a target protein due to the addition of a separate function, such as a binding site for other components. In the present invention, the term "CRP derivative" refers to a CRP wild-type protein from which a specific amino acid sequence has been removed or deleted, specifically, a C-terminal DNA binding motif has been removed, or AR1, AR2, and / or AR3 has been included, but is not limited thereto.

[0027] In the present invention, the CRP derivative is AR1 , CRP AR3 , CRP AR23 or CRP AR123 and more specifically, CRP AR123 However, the CRP is not limited to these. AR123is a wild-type CRPwt consisting of the sequence of SEQ ID NO:5, in which the C-terminal DNA-binding motif has been removed, and which contains the AR1, AR2 and AR3 domains and is composed of amino acids 1 to 180, specifically, the sequence of SEQ ID NO:6, but is not limited thereto.

[0028] In the present invention, the term "target gene" refers to a gene whose expression level is to be regulated by the introduction of a protein fusion. In the present invention, the target gene is a single gene or multiple genes, specifically, a gene composed of dxCas9-CRP or a -CRP derivative, but is not limited thereto. In the present invention, the term "target gene" is used interchangeably with "target gene".

[0029] In the present invention, "regulation of target gene expression" may refer to either enhancing or increasing the expression level of a target gene, or suppressing or decreasing the expression level, or may refer to a regulatory system capable of performing a dual function of simultaneously enhancing and suppressing the expression of a target gene. In the present invention, expression regulation is used interchangeably with expression control.

[0030] According to one embodiment of the present invention, the effect of enhancing the expression of a single target gene was confirmed. AR123 In the derivative-expressing strain, the amount of GFP expression increased by more than four times compared to the control strain (Figure 11), and in the expression inhibition effect experiment, it was confirmed that the amount of GFP expression was reduced by more than 84% compared to the control strain (Figure 12).

[0031] In addition, we confirmed the effects of enhancing and suppressing the expression of multiple genes, and found that dxCas9-CRP AR123 It was confirmed that the GFP expression level increased by approximately 13-fold or more compared to the control strain, and the mCherry expression level decreased by approximately 93% or more (Figure 13).

[0032] In the present invention, "guide RNA (gRNA)" refers to an RNA that contains a DNA base sequence of a target region and thereby specifically binds to a target gene and guides dxCas9 to the target site.

[0033] Typically, the guide RNA is a dual RNA containing two RNAs, i.e., crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA), as components, or a form containing a first portion containing a sequence that is fully or partially complementary to a sequence in the target DNA and a second portion containing a sequence that interacts with an RNA-guided nuclease. As an example, when the guide RNA is applied to Cpf1, the guide RNA may be crRNA. As another example, when the guide RNA is applied to Cas9, the guide RNA may be in the form of a dual RNA containing crRNA and tracrRNA as components, or in the form of a single-chain guide RNA (sgRNA) in which the main parts of crRNA and tracrRNA are fused together.

[0034] In the present invention, the gRNA is at least one gRNA selected from the group consisting of the base sequences of SEQ ID NOs: 23 to 25, but is not limited thereto.

[0035] In the present invention, the expression "having" a specific sequence includes including a sequence represented by the specific sequence, essentially consisting of the sequence, and consisting of the sequence. In the present invention, the sequence of the guide region is represented as one selected from the group consisting of the base sequences of SEQ ID NOs: 23 to 25, but the guide sequence of the present invention also includes a sequence in which a part of the guide region has been modified, for example, by addition, deletion, or substitution, as long as it has the ability to complementarily bind to a target DNA sequence.

[0036] In the present invention, the term "plasmid" refers to a collective term for a gene present in the cytoplasm of a bacterium and capable of replicating, or an extrachromosomal gene. The plasmid of the present invention contains one or more gRNAs containing a target gene sequence, and specifically expresses at least one gRNA selected from the group consisting of the base sequences of SEQ ID NOs: 23 to 25, but is not limited thereto.

[0037] Even if the present invention describes that a DNA, RNA, polynucleotide, gene, or protein has a base sequence or amino acid sequence of a specific sequence number, it goes without saying that a DNA, RNA, polynucleotide, gene, or protein having a sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added can also be used in the present invention, so long as it has the same or corresponding activity as the DNA, RNA, polynucleotide, gene, or protein consisting of the base sequence or amino acid sequence of that sequence number.

[0038] Furthermore, the present invention also includes sequences having 80% or more homology or identity with a base sequence or amino acid sequence represented by a specific SEQ ID NO, provided that they have the same or corresponding activity as the DNA, RNA, polynucleotide, gene, or protein of the base sequence or amino acid sequence represented by a specific SEQ ID NO., but is not limited thereto. Specifically, it goes without saying that the present invention also includes sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with the base sequence or amino acid sequence of a specific SEQ ID NO, and sequences that exhibit the corresponding efficacy or activity and have a partial deletion, modification, substitution, or addition of the sequence.

[0039] In the present invention, "homology" or "identity" means the degree to which two given amino acid sequences or nucleotide sequences are related to each other, and is expressed as a percentage.

[0040] Homology and identity are often used interchangeably.

[0041] Sequence homology or identity of conserved polynucleotides or polypeptides may be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize with at least about 50%, 60%, 70%, 80% or 90% or more of the entire sequence or full length under moderate or high stringent conditions. Hybridization may also be such that the polynucleotide has degenerate codons in place of codons.

[0042] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as in, for example, Non-Patent Document 1. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 3), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later), which includes the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, FASTA (Non-Patent Documents 5, 6 and 7). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0043] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 3, for example, as disclosed in Non-Patent Document 8. In summary, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include (1) a binary comparison matrix (identity takes a value of 1 and non-identity takes a value of 0) and a weighted comparison matrix of Non-Patent Document 10 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol of each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps. Thus, "homology" or "identity" in the present invention indicates the relevance between sequences.

[0044] In the present invention, "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing with each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the present invention may include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0045] Another aspect of the present invention provides a recombinant strain transformed with said plasmid.

[0046] The recombinant strain of the present invention is characterized by regulating gene expression, which includes, but is not limited to, enhancing or attenuating single or multiple target gene expression.

[0047] The "plasmid" and "target gene" are as described above.

[0048] In the present invention, "transformation" means expressing a protein encoded by a polynucleotide in a host cell by introducing a vector containing a polynucleotide encoding a target protein into the host cell. The transformed polynucleotide may be any polynucleotide that is expressed in the host cell, regardless of whether it is inserted into the chromosome of the host cell or located extrachromosomally. The polynucleotide may include DNA or RNA that encodes the target protein. The polynucleotide may be introduced in any form as long as it is introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements required for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

[0049] In addition, "operably linked" in the present invention means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target protein of the present invention.

[0050] The method for transforming the vector of the present invention may be any method for introducing a nucleic acid into a cell, and may be carried out by selecting a standard technique suitable for the host cell as known in the art, such as, but not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, etc.

[0051] In the present invention, the term "recombinant strain" refers to a strain into which a polynucleotide encoding a recombinant protein of interest has been introduced in the form of an expression vector or inserted into a chromosome so that the recombinant protein can be expressed and produced. Specifically, in the present invention, the recombinant strain is a strain in which not only a single target gene of the strain but also multiple genes have been enhanced and / or suppressed using dxCas9-CRP or a dxCas9-CRP derivative, but is not limited thereto.

[0052] In the present invention, the strain is of the genus Escherichia, specifically, Escherichia coli, but is not limited thereto.

[0053] Yet another aspect of the present invention provides a system for regulating target gene expression comprising dxCas9 and a CRP derivative.

[0054] The "dxCas9," "CRP derivative," "target gene," and "expression regulation" are as described above.

[0055] In the present invention, the system for regulating target gene expression refers to a system that enhances or suppresses the expression of a single target gene, or a system that enhances some genes and suppresses some genes in a multi-gene system, i.e., a system that can simultaneously enhance and suppress the expression of multiple genes, but is not limited to this.

[0056] In one embodiment of the present invention, it was confirmed that the GFP fluorescent expression of the target gene gRNA(A) was increased compared to the control group, and the mCherry fluorescent expression of the target gene gRNA(R2) was decreased compared to the control group. Therefore, it was confirmed that the system of the present invention enhances the expression of some genes and suppresses the expression of other genes in multiple genes, and it was found that gene expression enhancement and suppression regulation are possible simultaneously (Figure 13).

[0057] The present invention has a technical significance in that it has developed a target gene expression regulation system capable of activating and suppressing the transcription of a single gene or multiple genes.

[0058] Yet another aspect of the present invention provides a method for producing a recombinant strain that regulates target gene expression, comprising the steps of: i) preparing a dxCas9-CRP system in which a dxCas9 protein and a CRP protein are bound; ii) cloning a fluorescent reporter plasmid into the dxCas9-CRP system prepared as described above; iii) further cloning a guide RNA into the dxCas9-CRP system prepared in step ii); and iv) transforming the dxCas9-CRP-gRNA into a strain.

[0059] In the present invention, the guide RNA may include a target gene.

[0060] In the present invention, the dxCas9 is linked to a linker containing the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0061] In the present invention, the strain is of the genus Escherichia, specifically, but not limited to, Escherichia coli.

[0062] The "dxCas9", "CRP", "system", "guide RNA", "transformation", and "recombinant strain" are as described above.

[0063] In the present invention, the fluorescent reporter may be for verifying the performance of the dxCas9-CRP system. The fluorescent reporter may be, for example, luciferase, β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, enhanced yellow fluorescent protein (EYFP), emerald, CyPet, cyan fluorescent protein (CFP), cerulean, T-Sapphire, or alkaline phosphatase, specifically, but not limited to, GFP or mCherry.

[0064] Yet another aspect of the present invention provides a method for regulating target gene expression, comprising the step of applying a CRP derivative to dxCas9.

[0065] The "dxCas9", "CRP", "target gene", and "expression regulation" are as described above.

[0066] The present invention will be described in more detail below with reference to examples and experimental examples. However, these examples and experimental examples are merely illustrative of the present invention, and the present invention is not limited to these examples and experimental examples.

[0067] Experimental example 1. CRISPR-based gene expression regulation dxCas9-CRP AR123 System construction Experimental Example 1-1. Preparation of pdxCas9-linker Because overexpression of dxCas9 can cause cytotoxicity, we inserted the dxCas9 gene into the pAR-mlacI [rhaPBAD, p15A ori, CmR] vector and introduced an L-rhamnose inducible system, which allows precise expression regulation.

[0068] Specifically, to prepare dxCas9-linker consisting of dxCas9 (SEQ ID NO: 1) and linker (SEQ ID NO: 2), dxCas9-linker DNA was amplified by PCR using dxCas9(3.7)-VPR (Addgene plasmid #108383) plasmid as a template and primers of SEQ ID NO: 3 and 4. The primer sequences used in this experiment are shown in Table 1.

[0069] [Table 1]

[0070] Next, the pAR-mlacI vector cut with NdeI and SpeI restriction enzymes and the dxCas9-linker PCR product were subjected to homologous recombination to prepare a pdxCas9-linker plasmid, and the recombinant plasmid prepared as described above was transformed into E. coli DH5α strain by heat shock and cultured in solid LB medium containing 25 μg / ml of chloramphenicol antibiotic, and then the cells into which the recombinant plasmid was inserted were selected by PCR using the primers (SEQ ID NOs: 3 and 4). Then, the plasmid was extracted from the selected cells as described above, and finally the recombinant pdxCas9-linker plasmid was obtained.

[0071] Experimental Example 1-2. Preparation of expression plasmids for binding proteins of dxCas9 and CRP (WT and derivatives) CRP is structurally composed of AR1, AR2, and AR3 (activating region), which bind to the α-subunit of RNA polymerase to activate gene transcription, and binds to the CRP regulon genes via a DNA-binding motif at the C-terminus. In order to select the domain that binds to dxCas9 and functions as an effector to exert the greatest effect, we used the WT (wild type, CRP) of E. coli. WT , SEQ ID NO:5) CRP and four CRP derivatives, CRP AR123 (SEQ ID NO: 6), CRP AR23 (SEQ ID NO: 7), CRP AR1 (SEQ ID NO: 8) and CRP AR3 (SEQ ID NO: 9) was selected as the effector domain, and the gene expression regulating effect was confirmed.

[0072] Specifically, CRP WTis a wild-type transcriptional regulator of 45 kDa consisting of a total of 210 amino acids. At the N-terminus, there are activating regions AR2 (residues 20, 22, 97, and 102) and AR3 (residues 53-56 and 59) that bind to the α-subunit of RNA polymerase, and at the C-terminus, there are AR1 (residues 157-165) and a DNA binding motif (residues 182-194) that recognizes and binds to the CRP regulon genes. The N-terminus and C-terminus are connected by a short hinge region (residues 136-139). AR123 is the wild-type CRP WT The C-terminal DNA binding motif has been removed, and it contains all of the activating region (AR1, AR2, AR3) domains, and is composed of a total of 180 amino acids from the 1st amino acid to the 180th amino acid. AR23 is wild-type CRP WT CRP is composed of a total of 139 amino acids, including the AR2 and AR3 domains at the N-terminus and the hinge region, from the 1st amino acid to the 139th amino acid. AR1 contains only the C-terminal AR1 domain of wild-type CRP and consists of a total of 45 amino acids from amino acid 136 to amino acid 180. AR3 is wild-type CRP WT It contains only the N-terminal AR3 and is composed of a total of 60 amino acids from amino acid 28 to amino acid 92. These were selected and the experiment was carried out as follows.

[0073] In order to insert WT CRP and CRP derivatives into the pdxCas9-linker plasmid prepared in Experimental Example 1-1, primers were designed to partially express each domain of CRP using the genome of E. coli MG1655(DE3) as a template. WT are SEQ ID NOs: 10 and 13, CRP AR123 are SEQ ID NOs: 10 and 14, CRP AR23 are SEQ ID NOs: 10 and 15, CRP AR1 are SEQ ID NOs: 11 and 14, CRP AR3PCR was performed using primers of SEQ ID NO: 12 and SEQ ID NO: 16, respectively, and then each PCR product was obtained. The primer sequences used in the experiment are shown in Table 2.

[0074] [Table 2]

[0075] Next, the dxCas9-linker plasmid was treated with NgoMIV restriction enzyme and the amplified CRP derivative (CRP AR123 ,CRP AR23 ,CRP AR1 ,CRP AR3 ) pdxCas9-CRP WT , pdxCas9-CRP AR123 , pdxCas9-CRP AR23 , pdxCas9-CRP AR1 , pdxCas9-CRP AR3 The recombinant plasmid was then prepared. Next, the recombinant reaction product was transformed in the same manner as in Experimental Example 1-1, and the recombinant plasmid was selected using each of the primers (SEQ ID NOs: 10 to 16) used in amplifying the PCR products of the recombinant plasmid WT CRP and each of the CRP derivatives.

[0076] Experimental example 2. dxCas9-CRP AR123 Preparation of fluorescent reporter plasmids for system validation To test and verify the transcriptional regulation (enhancement / suppression) performance of the dxCas9-CRP system (Figures 1 and 2), the GFP fluorescent gene and the mCherry fluorescent gene, whose expression levels are easy to measure, were selected as reporter genes.

[0077] First, to verify the presence or absence of transcription enhancement, dxCas9-CRP must recognize and bind to the upstream region of the target gene. J23119 Or P J23117The sequence with RBS linked to it was obtained by gene synthesis. The synthetic gene obtained as described above was used as a template for PCR reaction, and J1-P J23119 -RBS is the primers of SEQ ID NOs: 17 and 18, J1-P J23117 The -RBS was prepared by PCR using primers of SEQ ID NO:19 and SEQ ID NO:20, and the pMW7-GFP plasmid was cleaved with NdeI restriction enzyme. The PCR products were then subjected to homologous recombination with the pMW7-GFP plasmid, in which the expression intensity of GFP was artificially regulated by a synthetic promoter. J23117 -GFP (weak GFP expression) and pMW7-P J23119 -GFP (strong GFP expression) recombinant plasmid was constructed (Figures 3 and 4).

[0078] In addition, to verify whether the dxCas9-CRP system can simultaneously regulate the transcriptional enhancement and suppression of multiple genes in a single cell, we artificially regulated the expression intensity of fluorescent genes using a synthetic promoter, and created pMW7-P, which induces persistent weak GFP expression and strong mCherry expression. J23117 -GFP-P J23119 A PCR was performed using primers of SEQ ID NO: 21 and 22 to generate the amplified P J23119 -mCherry PCR product and pMW7-P digested with AatII restriction enzyme J23117 Perform homologous recombination of -GFP plasmid into pMW7-P J23117 -GFP-P J23119 A plasmid with mCherry was constructed (Figure 5). The primers with SEQ ID NOs: 21 and 22 were used to construct the P J23119 Plasmids with -mCherry inserted were selected by PCR.

[0079] The primer sequences used to prepare the fluorescent reporter plasmids are shown in Table 4.

[0080] [Table 4]

[0081] Experimental Example 3. Selection of target sites for enhancing and suppressing fluorescent gene expression and preparation of gRNA The gRNA target position on the gene was selected so that dxCas9 would recognize and bind to the fluorescent gene of the fluorescent reporter plasmid prepared in Example 2, and further cloning for gRNA expression was performed into each pdxCas9-linker-CRP (or CRP derivative) plasmid prepared in Experimental Example 1-2.

[0082] Specifically, since gRNA contains the sequence of the target gene, and the transcription enhancement or suppression of the gene is determined depending on the position where gRNA binds to the target gene, the binding positions of gRNA(A) (SEQ ID NO: 23) for enhancing GFP expression and gRNA(R1) (SEQ ID NO: 24) for suppressing GFP expression, located at -191bp from the transcription start point adjacent to the PAM (5'-NGG-3') sequence, were selected, and the target position of gRNA(R2) (SEQ ID NO: 25) for suppressing mCherry expression, located at +28bp from the transcription start point of the mCherry gene, was selected (Figure 6). The gRNA sequence information used is shown in Table 5.

[0083] [Table 5]

[0084] Next, the gRNA is expressed and acts with a structure in which the sgRNA scaffold, which consists of a dxCas9 handle sequence with a 42 bp hairpin structure that commonly binds to dxCas9, and a 40 bp terminator derived from a Streptococcus pyogenes strain, is linked to the 20 bp sequence that binds complementary to the target gene. Therefore, all gRNAs show persistent and strong expression intensity. J23119 A promoter was introduced to induce overexpression (Figure 7).

[0085] To prepare pdxCas9-CRP-gRNA plasmids for expressing each gRNA, first, pgRNA-bacteria (Addgene #44251) for amplifying gRNA(A), gRNA(R1), and gRNA(R2) was used as a template, and PCR was performed using the primer combination of SEQ ID NO:26 and 27 and the primer combination of SEQ ID NO:28 and 33 for gRNA(A), the primer combination of SEQ ID NO:26 and 29 and the primer combination of SEQ ID NO:30 and 33 for gRNA(R1), and the primer combination of SEQ ID NO:26 and 31 and the primer combination of SEQ ID NO:32 and 33 for gRNA(R2), to obtain two PCR products. As described above, overlap PCR was performed based on the overlapping sequences of the two PCR products obtained, and PCR products of gRNA(A), gRNA(R1), and gRNA(R2) containing the target gene sequence were obtained. Next, the pdCas9-CRP (or CRP derivative) plasmid prepared in Experimental Example 1-2 was cleaved with SpeI and NotI restriction enzymes to obtain linear DNA, and the gRNA (A) and gRNA (R1) PCR products were subjected to a homologous recombination reaction to prepare recombinant pdxCas9-CRP (or CRP derivative)-gRNA (A) plasmid (FIG. 8, SEQ ID NO: 34) and pdxCas9-CRP (or CRP derivative)-gRNA (R1) plasmid (FIG. 9, SEQ ID NO: 35). The process of transforming the recombinant reaction product and selecting the recombinant plasmid was performed in the same manner as in Experimental Example 1-1, and the recombinant plasmid was selected by PCR using primers of SEQ ID NOs: 26 and 33. A recombinant plasmid in which gRNA (R2) was further inserted into pdxCas9-CRP-gRNA (A) was prepared by a homologous recombination reaction between the linear pdxCas9-CRP-gRNA (A) plasmid cleaved with the NotI restriction enzyme and the PCR product of gRNA (R2). The plasmid prepared as described above was then cleaved with NotI and NcoI restriction enzymes and subjected to agarose gel electrophoresis. The plasmid in which a DNA band corresponding to the size of gRNA(R2) was detected was selected to obtain the pdxCas9-CRP-gRNA(A / R2) plasmid (Figure 10, sequence number 36).

[0086] The primer sequences used in this experiment are shown in Table 6.

[0087] [Table 6]

[0088] [Table 7] EXAMPLES

[0089] Verification of gene expression enhancement / repression regulation by the dxCas9-CRP system Example 1-1. Confirmation of the effect of dxCas9-CRP in enhancing the expression of a single target gene and selection of a CRP derivative The recombinant pdxCas9-CRP (or CRP derivative)-gRNA (A) plasmid constructed in Experimental Example 3 and the GFP reporter plasmid constructed in Experimental Example 2 were transformed into E. coli MG1655 strain by the heat shock method, and then cultured in solid LB medium containing 25 μg / ml chloramphenicol and 100 μg / ml ampicillin to select the transformed strain. Next, the selected strain was cultured as described above in liquid LB medium containing 1 mM L-Rhamnose, and the cells cultured for 24 hours were photographed with a confocal fluorescence microscope, and then the GFP expression level of individual cells was measured by fluorescence intensity (Figure 11).

[0090] As a result, as shown in FIG. 11, in the control strain expressing dxCas9 and non-targeting gRNA, GFP fluorescence intensity was observed to be expressed at a relatively low level, and dxCas9-CRP AR123 It was confirmed that the amount of GFP fluorescence expression in the derivative-expressing strain was increased by more than four times compared to the control strain, and that this strain had the most excellent fluorescence expression compared to other CRP derivative-expressing strains.

[0091] Therefore, CRP AR123 The derivative was confirmed to have the greatest transcriptional enhancing effect as an effector of dxCas9, and CRP AR123The derivative contains all three activating regions, AR1, AR2, and AR3, that bind to RNA polymerase, and is therefore a CRP that exhibits excellent transcription enhancing regulatory activity. AR123 The derivative was selected as the most suitable CRP derivative and an experiment was carried out.

[0092] Example 1-2. Confirmation of the effect of dxCas9-CRP in suppressing single target gene expression and selection of CRP derivatives Since the dxCas9 protein highly efficiently inhibits transcription by inhibiting access of RNA polymerase when it binds to the promoter and ORF position of a target gene, experiments were conducted to confirm the effect of CRP (or a CRP derivative) on the gene transcription inhibition effect of dxCas9 itself when dxCas9 is fused to CRP (or a CRP derivative) and the efficiency of target gene expression inhibition of the dxCas9-CRP system.

[0093] Specifically, each recombinant pdxCas9-CRP (or CRP derivative)-gRNA (R1) plasmid constructed in Experimental Example 3 and the GFP reporter plasmid constructed in Experimental Example 2 were transformed into E. coli MG1655 strain by heat shock, and then cultured in solid LB medium containing 25 μg / ml chloramphenicol and 100 μg / ml ampicillin to select the transformed strain. Next, the selected strain was cultured as described above in liquid LB medium containing 1 mM L-Rhamnose, and the cells cultured for 24 hours were photographed with a confocal fluorescence microscope, and then the GFP expression level of individual cells was measured by fluorescence intensity (Figure 12).

[0094] As a result, as shown in FIG. 12, the strain expressing dxCas9 and gRNA(R1) simultaneously without CRP (or CRP derivative) showed a GFP fluorescence value reduced by about 75% or more compared to the control strain expressing dxCas9 and non-targeting gRNA, confirming that the gene of dxCas9 itself has a transcription repression effect. In particular, the dxCas9-CRP of the present invention AR123 It was confirmed that GFP expression was inhibited by approximately 84% or more in the derivative-expressing strain.

[0095] Therefore, the transcription inhibitory effect of dxCas9 due to the fusion of the CRP derivative was verified, and the gene expression enhancement and suppression experiments in Examples 1-1 and 1-2 showed that, among the CRP derivatives, CRP AR123 showed not only the highest transcriptional enhancing activity but also the highest repressing activity, and finally dxCas9-CRP AR123 It was confirmed that this is a new gene expression regulation system. EXAMPLES

[0096] dxCas9-CRP AR123 Simultaneous enhancement and inhibition of expression of multiple genes dxCas9-CRP selected in Examples 1-1 and 1-2 AR123 Using the system, an expression system was created to confirm the simultaneous expression enhancement and repression regulatory effects on multiple genes.

[0097] Specifically, pMW7-P constructed in Experimental Examples 2 and 3 J23117 -GFP-P J23119 -mCherry plasmid and pdxCas9-CRP AR123 The plasmid of -gRNA(A / R2) was co-expressed and selected in E. coli MG1655 in the same manner as in Example 1. Next, the selected E. coli cells were cultured for 24 hours in liquid LB medium containing 1 mM L-Rhamnose, 25 μg / ml chloramphenicol, and 100 μg / ml ampicillin antibiotic. The cultured cells were then harvested, suspended in 1X PBS (pH 8.0), and dispensed in 200 μL portions into 96-well plates. Next, the fluorescence intensity was measured using light in a wavelength range that can detect the fluorescence of GFP (488 / 509 nm) and mCherry (587 / 610 nm) in a microplate (FIG. 13).

[0098] dxCas9-CRP AR123 As a result of observing the fluorescence intensity of the strain expressing gRNA(A) and gRNA(R2), as shown in FIG. 13, dxCas9-CRP AR123The complex of dxCas9-CRP with gRNA(A) increased GFP fluorescence expression by approximately 12.6 times compared to the control strain. AR123 The complex of gRNA(R2) with mCherry reduced mCherry expression by approximately 93% compared to the control strain.

[0099] Therefore, dxCas9-CRP AR123 It was found that the system regulates the expression of a target gene that binds to the sequence of the gRNA complementary to the target gene. AR123 It was confirmed that the system can be used to target multiple cellular genes and simultaneously enhance and suppress the expression of multiple genes.

[0100] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

[0101] [Table 8-1]

[0102] [Table 8-2]

[0103] [Table 8-3]

[0104] [Table 8-4]

[0105] [Table 8-5]

[0106]

Table 8-6

[0107]

Table 8-7

[0108]

Table 8-8

[0109]

Table 8-9

[0110]

Table 8-10

[0111]

Table 8-11

[0112]

Table 8-12

[0113]

Table 8-13

[0114]

Table 8-14

[0115]

Table 8-15

[0116]

Table 8-16

[0117]

Table 8-17

Claims

1. A plasmid for regulating target gene expression comprising dxCas9 and a CRP derivative.

2. The CRP derivative is CRP ＡＲ１ , C.R.P. ＡＲ３ , C.R.P. ＡＲ２３ or CRP ＡＲ１２３ The plasmid for regulating expression of a target gene according to claim 1 ,

3. The CRP derivative is CRP ＡＲ１２３ The plasmid for regulating expression of a target gene according to claim 2 ,

4. The CRP ＡＲ１２３ is a wild-type CRP consisting of SEQ ID NO:5 ＷＴ 4. The plasmid for regulating target gene expression according to claim 3, which has a C-terminal DNA-binding motif removed, contains AR1, AR2 and AR3 domains and is composed of amino acids 1 to 180.

5. The CRP ＡＲ１２３ The plasmid for regulating target gene expression described in claim 3, wherein the derivative comprises the sequence of SEQ ID NO:

6.

6. The plasmid for regulating target gene expression described in claim 1, wherein the dxCas9 comprises the sequence of SEQ ID NO:

1.

7. The plasmid for regulating target gene expression according to claim 1 , wherein the expression regulation is to enhance or suppress expression of the target gene.

8. The plasmid for regulating target gene expression according to claim 1 , wherein the dxCas9 is linked to a linker comprising the amino acid sequence of SEQ ID NO:

2.

9. The plasmid for regulating target gene expression according to claim 1 , wherein the plasmid comprises at least one guide RNA (gRNA) comprising a target gene sequence.

10. The plasmid for regulating target gene expression according to claim 9, wherein the guide RNA is at least one selected from the group consisting of the base sequences of SEQ ID NOs: 23 to 25.

11. A recombinant strain transformed with the plasmid according to any one of claims 1 to 10.

12. The recombinant strain of claim 11, wherein the strain regulates gene expression.

13. The recombinant strain of claim 11 , wherein the strain enhances or represses single or multiple target gene expression.

14. The recombinant strain of claim 11 , wherein the strain is of the Escherichia genus.

15. The recombinant strain of claim 11, wherein the strain is Escherichia coli.

16. A system for regulating target gene expression comprising dxCas9 and a CRP derivative.

17. i) preparing a dxCas9-CRP system in which a dxCas9 protein and a CRP protein are bound; ii) cloning a fluorescent reporter plasmid into the dxCas9-CRP system prepared as described above; iii) Further cloning a guide RNA into the dxCas9-CRP system prepared in step ii); iv) transforming the dxCas9-CRP-gRNA into a strain.

18. The method for producing a recombinant strain according to claim 17, wherein the guide RNA comprises a target gene.

19. The method for producing a recombinant strain according to claim 17, wherein the dxCas9 is linked to a linker comprising the amino acid sequence of SEQ ID NO:

2.

20. The method for producing a recombinant strain according to claim 17, wherein the strain is of the Escherichia genus.

21. The method for producing a recombinant strain according to claim 17, wherein the strain is Escherichia coli.

22. 18. The method for producing a recombinant strain according to claim 17, wherein the fluorescent reporter is selected from the group consisting of luciferase, β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, enhanced yellow fluorescent protein (EYFP), emerald, CyPet, cyan fluorescent protein (CFP), cerulean, T-Sapphire, and alkaline phosphatase.

23. A method for regulating target gene expression, comprising a step of applying a CRP derivative to dxCas9.

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