KR20210078894A - Compositon for inhibiting gene expression using CRISPRi - Google Patents

Abstract

The present invention relates to a CRISPR-dCas12a vector system capable of inhibiting gene expression using CRISPR interference. By targeting cyanobacteria, the present invention can be used to control the metabolic flow of substances useful for high-efficiency photoconversion of carbon dioxide to reduce and remove carbon dioxide in an environmentally friendly manner. Compared to the conventional gene expression system, there is less time constraint and the present invention has excellent economic feasibility. Since toxicity is reduced in cyanobacteria, the present invention has advantageous properties for productivity, and has the effect of being able to be used for metabolic engineering research.

Description

Composition for inhibiting gene expression using CRISPR interference {Compositon for inhibiting gene expression using CRISPRi}

The present invention relates to gene expression suppression using CRISPR interference, and more particularly, to a CRISPR-dCas12a vector system capable of suppressing gene expression in cyanobacteria.

Currently, in the bio-industry, a technique for efficiently producing a product that cannot be produced by a wild type by metabolic engineering of an industrial microorganism to produce a high value-added product is widely used. The key to these technologies is to efficiently and economically produce the target material. Metabolic engineering methods used at this time include techniques for inserting heterologous genes into plasmids or genomes or making mutations of specific genes. In particular, recently, a technology capable of rapidly obtaining insertion or mutation of a target gene using CRISPR has been in the spotlight.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPRassociated (Cas) proteins are involved in the adaptive immune system in Eubacteria and Archaea. The CRISPR-Cas system has been developed and used as a target gene editing tool in living organisms including humans. CRISPR can easily and quickly create gene insertion and modification at a target site using Cas and sgRNA, but CRISPR has the potential to cause mutations at sites other than the target site. Therefore, CRISPRi has recently been used as a more advanced technology. CRISPR interference (CRISPRi) is to use the CRISPR-Cas system for the purpose of regulating gene expression instead of editing the target gene, and suppressing the gene at the target location using dCas that lacks (removes) the DNA-cleaving enzyme activity of Cas. It is a system that can lead to The advantage of this technology is that it can suppress gene expression at the target site without making mutations, so it is possible to control genes without causing genetic modification, and it is being used as an efficient tool for DNA sequence-specific regulation of gene expression in various living organisms.

Cas9 (SpdCas9), in which the DNA cleavage activity of Streptococcus pyogenes derived from the Type II CRISPR system has been removed, is the most studied and most widely used protein in CRISPRi. The CRISPRi system works with SpdCas9 protein, CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA). The SpCas9-crRNA-tracrRNA complex binds to the nontemplate strand of the target DNA and inhibits transcription by RNA polymerase. In addition, a multimeric CRISPRi system derived from the Type I CRISPR system that requires deletion of the Cas3 protein involved in cleavage and degradation of target DNA has also been reported. The CRISPRi systems derived from Type I and II CRISPR systems can inhibit gene transcription efficiently, reversibly and multiplely, but are suitable for applications in that they require tracrRNA in addition to specific protospacer adjacent motif (PAM) sequences and crRNAs. There are limits.

Recently, the Type V-A CRISPR system has been shown to be a targeted genome editing tool in human cells. It consists of a single Cpf1 (CRISPR from Prevotella and Francisella 1) protein and its cognate crRNA and works without the need for an additional tracrRNA. In contrast to Cas9, which recognizes a guanidine-rich PAM sequence downstream of the target DNA, Cpf1 recognizes a thymidine-rich PAM sequence upstream of the target DNA. do. In addition, Cpf1 cuts the target DNA to generate a staggered end, but Cas9 makes a blunt end, so it can be used in areas where it is difficult to apply a Cas protein-based system. Type V-A CRISPR-Cpf1 is an attractive alternative to Cas9-based genome editing tools. To date, the PAM sequence and DNA binding activity of Francisella novicida U112 (FndCpf1), a Cpf1 whose DNA cleavage activity has been removed, has been reported. However, although the diversity of Cpf1 family proteins has been studied by searching the public sequence database recently, only the PAM sequences of a few Cpf1 family proteins have been identified, and CRISPRi has not been developed. Korean Patent Application Laid-Open No. 10-2015-0107739 relates to altering the expression of a gene product using the CRISPR system, but since it uses a Cas9 protein, it has the disadvantages of the above-described Cas-based system as it is.

Recent advances in genome sequencing techniques and analytical methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. Precise genomic targeting techniques are being developed to advance synthetic biology, biotechnology, and medical applications, as well as to allow systematic reverse engineering of causal genetic variations by allowing for the selective perturbation of individual genetic elements. need. Genome-editing techniques, such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases, are used to generate targeted genomic alterations. Although available, there remains a need for novel genome engineering techniques that use novel strategies and molecular mechanisms, are available, easy to set up, scalable, and capable of targeting multiple locations within the eukaryotic genome.

Recently, due to the ripple effect of CRISPR technology, interest in the future market is increasing. In addition to disease treatment, as CRISPR technology, the market size of CRISPR is increasing over time in genome editing, genetic engineering, gene library, genetically modified food, CRISPR plasmid, human stem cell, cell line engineering, etc. R&D is being actively carried out in order to commercialize technology and preoccupy the advantage. Accordingly, while various application studies for Cas9 CRISPR technology are being conducted from animal cells to microorganisms, research on approaches suitable for cyanobacteria are also being actively conducted. As an example, based on the difference between Cas9 and Cas12a proteins, Synechocystis sp. A study has been reported to monitor gene regulation expression and survival of the strain in PCC 6803. In this trend, it is urgent to develop a gene tool such as a CRISPR gene expression control system that can control the flow of metabolic engineering to maximize the use and efficiency of carbon dioxide photoconversion. With the active development of synthetic biology technology for useful substances through carbon dioxide immobilization based on photosynthetic ability using solar energy, efficient photoconversion biological access of cyanobacteria capable of genetic recombination is an economical method that can occur in the culture of microbial strains other than photosynthesis. It is a technology that can overcome limitations. While the study of the molecular mechanism of metabolic flow in cyanobacteria has not been clearly identified, the application of metabolic engineering and systems biology techniques is highly required. In addition, the existing gene introduction method in cyanobacteria takes a long time and has a low efficiency of introducing foreign genes, and the Cas9 CRISPR protein gene regulation system has a growth inhibition risk in the study of essential growth genes of cyanobacteria. . In addition, it has been reported that the Cas9 protein CRISPR system is toxic to cyanobacterial strains.

It is an object of the present invention to provide a composition for inhibiting gene expression.

Another object of the present invention is to provide a recombinant vector for suppressing gene expression.

Another object of the present invention is to provide a kit for suppressing gene expression.

In addition, it is an object of the present invention to provide a method for inhibiting the expression of a target gene.

In order to solve the above problems, the present invention provides a composition for inhibiting gene expression comprising dCas12a and crRNA.

In addition, the present invention provides a recombinant vector for suppressing gene expression comprising a nucleic acid sequence encoding dCas12a, a nucleic acid sequence encoding crRNA, and a promoter.

In addition, the present invention provides a kit for inhibiting gene expression comprising the composition or vector.

In addition, the present invention provides a method for inhibiting the expression of a target gene comprising the step of transforming the vector.

The use of the CRISPR-dCas12a vector system of the present invention for inhibiting gene expression through CRISPR interference is a cyanobacterial target system, so it regulates the metabolic flow of substances useful for high-efficiency photoconversion of carbon dioxide to reduce and remove carbon dioxide in an environmentally friendly manner It can be used for this purpose, has less time constraint compared to the conventional gene expression system, has excellent economic feasibility, and has advantageous properties for productivity because toxicity in cyanobacteria is reduced, so it has the effect of being able to utilize it for metabolic engineering research.

1 is a diagram showing a schematic diagram of a cyanobacterial gene expression control vector of the present invention.
2 is a view confirming the degree of inhibition of the expression of a target gene in a cyanobacterial strain according to the length (19nt, 23nt or 36nt) of the DR (direct repeat) in the crRNA of the vector of the present invention.
3 is a diagram confirming the degree of inhibition of the expression of a target gene in a cyanobacterial strain according to the direction (T strand direction or NT strand direction) of the protospacer in the crRNA of the vector of the present invention.
Figure 4 is a diagram confirming the CRISPR dCas12a system implementation of the vector of the present invention through the phenotype of cyanobacteria:
a: Schematic diagram of the mechanism of inhibition of target gene expression of CRISPR dCas12a protein;
b: phenotype according to the expression level of the nbl A gene; and
c: mRNA expression level of dCas12a.
5 is a diagram confirming the multi-gene expression inhibitory effect of the vector of the present invention:
a: A schematic diagram of a multi-target gene array in a vector;
b: phenotype according to the expression level of the nbl A gene;
c: degree of inhibition of eyfp gene expression as determined by fluorescence intensity; and
d: The degree of inhibition of gfp gene expression as determined by fluorescence intensity.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

In addition, the terms used in this specification are terms used to properly express the preferred embodiment of the present invention, which may vary according to the intention of a user or operator, or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

In one aspect, the present invention is a DNA cleavage activity is inactivated (nuclease-deactivated) Cas12a protein (dCas12a); And it relates to a composition for suppressing gene expression comprising a crRNA (CRISPR RNA) comprising a repetitive nucleotide sequence (direct repeat, DR) and a protospacer.

In one embodiment, dCas12a may comprise the amino acid of SEQ ID NO: 1.

In one embodiment, the repetitive nucleotide sequence may be 10 nt to 46 nt in length, more preferably 15 to 22 nt in length, and most preferably 19 nt in length.

In one embodiment, it may be a protospacer in the T strand direction of the template DNA.

In one embodiment, the composition for suppressing gene expression may be a composition for suppressing gene expression of cyanobacteria, and the cyanobacteria may be Synechococcus elongatus.

In one embodiment, in the crRNA, two or more different repetitive nucleotide sequences and protospacers may be sequentially repeated, the protospacers may be complementary to two or more different target genes, and multiple gene expression may be simultaneously inhibited. have.

In the present invention, by repeatedly linking a protospacer, which is a nucleic acid that hybridizes with a repetitive nucleotide sequence (direct repeat, DR) and a target DNA, and modifying each repetitive nucleotide sequence and/or protospacer to suit the desired gene. It is possible to construct crRNA that suppresses multiple genes. The crRNA may hybridize with the target DNA.

As used herein, the term “protospacer” refers to a nucleic acid sequence that hybridizes with a target DNA.

As used herein, the term "hybridization" refers to a reaction in which one or more nucleic acids react to form a complex, and the complex is stabilized through hydrogen bonding between bases of nucleic acid residues.

As used herein, the term "expression" refers to the process by which a nucleic acid is transcribed from a DNA template (eg, into mRNA or other RNA transcript) and/or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. refers to

As used herein, the term “cleavage” refers to breakage of a covalent backbone of a nucleic acid molecule.

The composition for inhibiting gene expression of the present invention is in the form of a recombinant vector containing DNA encoding a Cas12a protein (dCas12a) in which DNA cleavage activity is inactivated (nuclease-deactivated) and a recombinant vector containing DNA encoding crRNA cells. Alternatively, it may be introduced into an organism, introduced into a cell or organism in the form of a mixture comprising dCas12a protein and crRNA or a ribonucleic acid protein forming a complex thereof, or may be introduced into a cell or organism as a vector including dCas12a protein and crRNA together.

In the present invention, the specific sequence of the crRNA may be appropriately selected according to the type of Cas12a protein (derived microorganism).

In the present invention, the endonuclease such as the Cas12a protein may be isolated from a microorganism or non-naturally occurring by a recombinant method or a synthetic method. The endonuclease may further include an element commonly used for intranuclear delivery of cells at the N-terminus or C-terminus (or at the 5' end or 3' end of the nucleic acid molecule encoding it). , but not limited thereto. The endonuclease protein may be used in the form of a purified protein, DNA encoding it, or a recombinant vector containing the DNA.

It may further include a protospacer adjacent motif (PAM) consisting of TTN (A or C) upstream of the target DNA.

As used herein, the term "upstream" refers to a sequence located in the 5' direction with respect to a specific position of the nucleotide sequence, and "downstream" is based on a specific position of the nucleotide sequence, It refers to a sequence located in the 3' direction.

In one aspect, the present invention provides a nucleic acid sequence encoding a Cas12a protein (dCas12a) having inactivated DNA cleavage activity; a nucleic acid sequence encoding a crRNA including a repetitive nucleotide sequence and a protospacer; And it relates to a recombinant vector for suppressing gene expression comprising a promoter operably linked to the nucleic acid sequence.

In one embodiment, the nucleic acid sequence encoding dCas12a may include the nucleotide sequence of SEQ ID NO: 2.

In one embodiment, the crRNA may simultaneously inhibit multiple gene expression by sequentially repeating two or more different repetitive nucleotide sequences and protospacers.

In one embodiment, the vector of the present invention is a recombinant vector for inhibiting gene expression for cyanobacteria

In one embodiment, the repetitive nucleotide sequence may be 10 nt to 46 nt in length, more preferably 19 nt in length.

In one embodiment, it may be a protospacer in the T strand direction of the template DNA.

In the present invention, a nucleic acid sequence may be "exogenous", indicating that it is heterologous to the bacterium into which the vector is introduced, or that the sequence is homologous to the bacterium's sequence, but is located within a bacterial cell nucleic acid in which the sequence is not normally expressed. it means. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (eg, YACs). Those skilled in the art will be well able to construct vectors via standard recombinant techniques (see, eg, Maniatis et al, 1989 and Ausubel et al, 1994, incorporated herein by reference).

As used herein, the term "expression vector" refers to any type of genetic construct comprising a nucleic acid encoding an RNA capable of being transcribed. In some cases, RNA molecules are translated into proteins, polypeptides or peptides. In still other cases, these sequences are not translated, for example, upon generation of antisense molecules or ribosomes. Expression vectors may contain a variety of "regulatory sequences", which are nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular bacterial cell. In addition to the regulatory sequences that govern transcription and translation, vectors and expression vectors perform other functions and may contain the nucleic acid sequences described below.

As noted above, the nucleic acid encoding the desired product to be expressed can be operably linked to appropriate expression control sequences for each bacterial cell. It can include regulatory sequences such as those described throughout this specification, ribosome binding sites, and transcription termination signals.

As used herein, the term "promoter" is a regulatory sequence that is a region of a nucleic acid sequence that regulates the initiation and rate of transcription. It may contain genetic elements to which regulatory proteins and molecules such as RNA polymerase and other transcription factors can be bound to initiate specific transcription of a nucleic acid sequence. The phrases “operably located,” “operably linked,” “under control,” and “under transcriptional control,” refer to a promoter having an appropriate site of action in relation to a nucleic acid sequence for regulating the transcriptional initiation and/or expression of the nucleic acid sequence. and/or in a direction. Promoters generally include sequences that function to localize the starting site for RNA synthesis. Additional promoter elements control the frequency of transcription initiation. Typically, it is located in a region up to 100 bp from the start site, but it has been found that many promoters also contain functional elements downstream of the start site. In order for the coding sequence to be "under the control" of a promoter, the 5' end of the transcription initiation site of the transcriptional reading frame is located "downstream" (ie, 3') of the selected promoter. An “upstream” promoter stimulates transcription of DNA and expression of the encoded RNA. The spacing between promoter elements can vary so much that promoter function is preserved even when the elements are retrograde or move relative to each other. Depending on the promoter, it is self-evident that the individual elements act jointly or individually to activate transcription. A promoter may or may not be used in conjunction with an "enhancer", which represents a cis-acting regulatory sequence that is involved in the transcriptional activation of a nucleic acid sequence. A promoter may be naturally associated with a nucleic acid sequence, which may be obtained by isolating a 5' non-coding sequence located upstream of a coding segment and/or an exon. Such promoters may be referred to as “endogenous”. Similarly, an enhancer may be naturally associated with a nucleic acid sequence located downstream or upstream of the nucleic acid sequence. Alternatively, certain advantages may be achieved by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter not normally associated with the nucleic acid sequence under its natural environment. A recombinant or heterologous enhancer, operator or other regulatory sequence also refers to an enhancer, operator or other regulatory sequence not normally associated with a nucleic acid sequence in its natural environment. Such promoters, enhancers, operators, or other regulatory sequences may include promoters, enhancers, operators or other regulatory sequences of other genes, any other virus, or promoter, enhancer, operator or other regulatory sequence isolated from any other virus or prokaryotic or eukaryotic cell. regulatory sequences, and promoters, enhancers, operators or other regulatory sequences that are not "naturally occurring", ie, contain different elements and/or variations of different transcriptional regulatory regions that alter expression.

In the present invention, cells containing the vector of the present invention can be identified in vitro or in vivo by containing a marker in the expression vector. Such markers will confer an identifiable change to the cell to facilitate identification of a cell containing the expression vector. In general, a selection marker is one that imparts a property that permits selection. A positive selection marker is one that allows its selection in the presence of the marker, whereas a negative selection marker is one that prevents its selection due to its presence. An example of a positive selection marker is a drug resistance marker. In general, introduction of drug selection markers aids in the cloning and identification of transformants, e.g., resistance to chloramphenicol, erythromycin, gentimycin, spectinomycin, streptomycin, zeocin and kanamycin. Contributing genes are useful selection markers. Complementary methods in which an auxotroph is functionally complemented by a deficient gene are also used. In addition to markers conferring phenotypes that allow for the distinction of transformants based on performance conditions, other types of markers are also contemplated, including screening markers such as GFP and YFP based on colorimetric fluorescence analysis. Also, a marker using luciferase can be used as a reporter gene. The skilled artisan will also know how to use immunological markers, possibly in conjunction with FACS analysis. The marker used is not so critical as long as it can be expressed simultaneously with the nucleic acid encoding the gene product. In addition, examples of selection and screening markers are well known to those skilled in the art.

In one aspect, the present invention relates to a kit for inhibiting gene expression of cyanobacteria comprising the vector of the present invention.

In one aspect, the present invention comprises the steps of constructing the vector of claim 7 comprising a protospacer complementary to the target gene; And it relates to a method for inhibiting the expression of a target gene in cyanobacteria, comprising the step of transforming the vector into cyanobacteria.

In one embodiment, the vector may suppress the expression of one or more different target genes by including a crRNA in which a protospacer is sequentially repeated at least two repetitive base sequences and a protospacer complementary to one or more different target genes.

The transformation according to the present invention includes any method of introducing a desired gene (nucleic acid) into an organism, cell, tissue or organ, and as is known in the art, it can be performed by selecting an appropriate standard technique according to the host cell. can These methods include electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation using silicon carbide fibers, agrobacterium mediated transformation, PEG, dextran sulfate, lipofect. Tamine, thermal shock, and the like are included, but are not limited thereto.

In one aspect, the present invention relates to a method for inhibiting the expression of a target gene in a cell or organism, comprising the step of treating the cell or organism with the composition for inhibiting gene expression of the present invention.

In one embodiment, the cell may be a prokaryotic cell, and may be a cyanobacteria.

The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the contents of the present invention, and the present invention is not limited thereto.

Example 1. Cyanobacterial gene expression control vector system construction

The pSe3Bblc-GFP vector capable of gene expression under the operation of the pTrc promoter was treated with EcoRI and XhoI, and then a codon-optimized dCas12a protein-coding gene fragment treated with BglII and XhoI was ligated and inserted (pSe3Bb1c-dCas12a). Then, BglII and XhoI-treated crRNA (J23119, leader sequence, direct repeat, eyfp protospacer P1 and terminator) and eyfpDNA fragment were sequentially inserted into pSe3Bb1c-dCas12a treated with BamHI and XhoI using T4 ligase. The vector Syne dCas12a-guide crRNA E. coli DH10B pSyne7942-dCas12a-crRNA-eYFP was constructed ( FIG. 1 ).

Example 2. Target gene expression inhibition optimization crRNA length selection

Syne dCas12a-guide crRNA E. coli DH10B pSyne7942 as in Example 1 to include crRNA each having a DR of 19nt, 23nt or 36nt in order to confirm the degree of gene expression inhibition according to the DR (direct repeat) length of crRNA. -dCas12a-crRNA-eYFP vector was prepared, and each of them was introduced into cyanobacteria (Synechococcus elongatus ) to prepare a transformed strain. As a result of comparing the fluorescence intensity of eYFP and the mRNA expression level (transcription level) of dCas12a in the three types of transformed strains, the strain transformed with a vector containing crRNA each having a DR of 19nt, 23nt or 36nt length was The fluorescence intensity of the strain expressing only the dCas12a protein was reduced by 78%, 63.7%, and 49.3%, respectively, and the mRNA expression level of dCas12a was also reduced by 90%, 80%, and 80%, respectively, so that the crRNA that most significantly inhibits the expression of the target gene The length condition was found to be 19 nt (Fig. 2).

Example 3. Target gene expression inhibition optimization crRNA directional selection

The direction of the protospacer (23bp sequence at the end of the recognition sequence PAM 5-TTN-3' of dCas12a and the target gene) essential for forming the CRISPR complex of the reporter genes eYFP, dCas12a and crRNA on the cyanobacterial genome (template DNA) As a result of comparing the expression inhibitory effect of the target gene according to the T strand or NT strand of the transgenic strain through the fluorescence intensity of eYFP and the mRNA expression level of dCas12a, compared to the fluorescence intensity of the strain expressing only the dCas12a protein, the T strand ( strand) decreased by 61.9%, and in the case of NT strand, it decreased by 29.7%. In addition, the mRNA expression level of dCas12a also decreased by 75.5% and 53%, respectively, indicating that when the crRNA protospacer was in the T-strand direction, the inhibition of gene expression in Synecocos elongatus was significantly inhibited compared to the NT-strand direction (Fig. 3). ).

Example 4. Confirmation of CRISPR dCas12a system implementation through cyanobacterial phenotype

The phycobilisome protein complex, a light harvest pigment in the thylakoid membrane in Synecococcus elongatus strain, is an important material for photosynthesis, but in an environment lacking nitrogen (N), it decomposes the pigments to produce N To obtain phycobilisome protein degradation protease (coding gene: nbl A ) is increased. As such, when the expression of the nbl A gene is increased, the cyanobacterial phenotype changes to bright yellow (chlorosis, clorosis). Therefore, in order to confirm whether the CRISPR dCas12a system using the vector of the present invention is well implemented, the phenotype of the strain transformed with the vector containing the CRISPR dCas12a-guide crRNA P2 or P2' targeting nbl A is dCas12a It appears more green than the strain or wild-type phenotype containing only the protein (Fig. 4b), indicating that the CRISPR dCas12a system of the present invention inhibits the color change (whitening phenomenon) of cyanobacteria. In addition, the mRNA expression level of dCas12a was also decreased by 98.2% (Fig. 4c).

Example 5. Multi-gene expression inhibition confirmation

In order to confirm that the vector of the present invention is capable of multi-gene expression control, the protospacer was used to target the eyfp (P1), nblA (P2) and gfp (P3) genes in the crRNA region of the vector prepared in Example 1 above. Array 1 is eyfp, array 2 is eyfp and nblA, and array 3 is made to contain the target gene protospacers of eyfp, nblA and gfp (FIG. 5a) After time incubation, IPTG was induced to operate the dCas12a-crRNA system, and the degree of inhibition of target gene expression of each strain was confirmed by fluorescence intensity and phenotype (in the case of nblA gene).

As a result, in the case of the phenotype (nblA crRNA P2), it was green compared to the strain containing only dCas12a protein or the wild-type strain under the N-deficient condition. appeared (Fig. 5b). In addition, eyfp (eyfp crRNA P1) included in arrays 1 to 3 also showed a 62.4%, 76.3% and 76.2% decrease in fluorescence intensity compared to the strain containing only the dCas12a protein or the wild-type strain, respectively. was confirmed to work (Fig. 5c). In addition, as a result of confirming the inhibition of gfp gene expression by gfp crRNA P3 included in array 3 by fluorescence, the fluorescence intensity was decreased by 81.3% compared to the strain expressing only the dCas12a protein ( FIG. 5d ). Through this, it can be seen that the crRNA present in the array form inhibits the expression of multi-target genes on the cyanobacterial genome.

<110> Research and Business Foundation SUNGKYUNKWAN UNIVERSITY <120> Compositon for inhibiting gene expression using CRISPRi <130> R-2019-0646-EN-1 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 1300 <212> PRT <213> Artificial Sequence <220> <223> dCas12a <400> 1 Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr 1 5 10 15 Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys 20 25 30 Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys 35 40 45 Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu 50 55 60 Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser 65 70 75 80 Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys 85 90 95 Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr 100 105 110 Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile 115 120 125 Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln 130 135 140 Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr 145 150 155 160 Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr 165 170 175 Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser 180 185 190 Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu 195 200 205 Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys 210 215 220 Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu 225 230 235 240 Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg 245 250 255 Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr 260 265 270 Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys 275 280 285 Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile 290 295 300 Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys 305 310 315 320 Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser 325 330 335 Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met 340 345 350 Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys 355 360 365 Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln 370 375 380 Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr 385 390 395 400 Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala 405 410 415 Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn 420 425 430 Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala 435 440 445 Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn 450 455 460 Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala 465 470 475 480 Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys 485 490 495 Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys 500 505 510 Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp 515 520 525 Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His 530 535 540 Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His 545 550 555 560 Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val 565 570 575 Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser 580 585 590 Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly 595 600 605 Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys 610 615 620 Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile 625 630 635 640 Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys 645 650 655 Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val 660 665 670 Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile 675 680 685 Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln 690 695 700 Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe 705 710 715 720 Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp 725 730 735 Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu 740 745 750 Phe Tyr Arg Glu Val Glu Asn Gin Gly Tyr Lys Leu Thr Phe Glu Asn 755 760 765 Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr 770 775 780 Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg 785 790 795 800 Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn 805 810 815 Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr 820 825 830 Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala 835 840 845 Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu 850 855 860 Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe 865 870 875 880 His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe 885 890 895 Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His 900 905 910 Ile Leu Ser Ile Ala Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu 915 920 925 Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile 930 935 940 Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile 945 950 955 960 Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn 965 970 975 Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile 980 985 990 Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu 995 1000 1005 Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr 1010 1015 1020 Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe 1025 1030 1035 1040 Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln 1045 1050 1055 Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly 1060 1065 1070 Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val 1075 1080 1085 Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys 1090 1095 1100 Ser Gln Glu Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp 1105 1110 1115 1120 Lys Gly Tyr Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys 1125 1130 1135 Ala Ala Lys Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile 1140 1145 1150 Asn Phe Arg Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val 1155 1160 1165 Tyr Pro Thr Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu 1170 1175 1180 Tyr Gly His Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp 1185 1190 1195 1200 Lys Lys Phe Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln 1205 1210 1215 Met Arg Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro 1220 1225 1230 Val Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys 1235 1240 1245 Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu 1250 1255 1260 Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys 1265 1270 1275 1280 Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln 1285 1290 1295 Asn Arg Asn Asn 1300 <210> 2 <211> 3903 <212> DNA <213> Artificial Sequence <220> <223> dCas12a <400> 2 atgtctatat atcaagaatt tgtgaataaa tattctcttt ctaaaacgct tcgttttgaa 60 cttataccgc aagggaaaac gcttgaaaat ataaaagcgc gtgggcttat acttgatgat 120 gaaaaacgtg cgaaagatta taaaaaagcg aaacaaataa tagataaata tcatcaattt 180 tttatagaag aaatactttc ttctgtgtgt atatctgaag atttgcttca aaattattct 240 gatgtgtatt ttaaacttaa aaaatctgat gatgataatc ttcaaaaaga ttttaaatct 300 gcgaaagata cgataaaaaa acaaatatct gaatatataa aagattctga aaaatttaaa 360 aatcttttta atcaaaatct tatagatgcg aaaaaagggc aagaatctga tcttatactt 420 tggcttaaac aatctaaaga taatgggata gaacttttta aagcgaatag cgatataacg 480 gatatagatg aagcgcttga aataataaaa tcttttaaag ggtggacgac gtattttaaa 540 gggtttcatg aaaatcgtaa aaatgtgtat tcttctaatg atataccgac gtctataata 600 tatcgtatag tggatgataa tcttccgaaa tttcttgaaa ataaagcgaa atatgaatct 660 cttaaagata aagcgccgga agcgataaat tatgaacaaa taaaaaaaga tttggcggaa 720 gaacttacgt ttgatataga ttataaaacg tctgaagtga atcaacgtgt gttttctctt 780 gatgaagtgt ttgaaatagc gaattttaat aattatctta atcaatctgg gataacgaaa 840 tttaatacga taataggggg gaaatttgtg aatggggaaa atacgaaacg taaagggata 900 aatgaatata taaatcttta ttctcaacaa ataaatgata aaacgcttaa aaaatataaa 960 atgtctgtgc tttttaaaca aatactttct gatacggaat ctaaatcttt tgtgatagat 1020 aaacttgaag atgattctga tgtggtgacg acgatgcaat ctttttatga acaaatagcg 1080 gcgtttaaaa cggtggaaga aaaatctata aaagaaacgc tttctcttct ttttgatgat 1140 cttaaagcgc aaaaacttga tctttctaaa atatatttta aaaatgataa atctcttacg 1200 gatctttctc aacaagtgtt tgatgattat tctgtgatag ggacggcggt gcttgaatat 1260 ataacgcaac aaatagcgcc gaaaaatctt gataatccgt ctaaaaaaga acaagaactt 1320 atagcgaaaa aaacggaaaa agcgaaatat ctttctcttg aaacgataaa acttgcgctt 1380 gaagaattta ataaacatcg tgatatagat aaacaatgtc gttttgaaga aatacttgcg 1440 aattttgcgg cgataccgat gatatttgat gaaatagcgc aaaataaaga taatcttgcg 1500 caaatatcta taaaatatca aaatcaaggg aaaaaagatt tgcttcaagc gtctgcggaa 1560 gatgatgtga aagcgataaa agatttgctt gatcaaacga ataatcttct tcataaactt 1620 aaaatatttc atatatctca atctgaagat aaagcgaata tacttgataa agatgaacat 1680 ttttatcttg tgtttgaaga atgttatttt gaacttgcga atatagtgcc gctttataat 1740 aaaatacgta attatataac gcaaaaaccg tattctgatg aaaaatttaa acttaatttt 1800 gaaaattcta cgcttgcgaa tgggtgggat aaaaataaag aaccggataa tacggcgata 1860 ctttttataa aagatgataa atattatctt ggggtgatga ataaaaaaaa taataaaata 1920 tttgatgata aagcgataaa agaaaataaa ggggaagggt ataaaaaaat agtgtataaa 1980 cttcttccgg gggcgaataa aatgcttccg aaagtgtttt tttctgcgaa atctataaaa 2040 ttttataatc cgtctgaaga tatacttcgt atacgtaatc attctacgca tacgaaaaat 2100 gggtctccgc aaaaagggta tgaaaaattt gaatttaata tagaagattg tcgtaaattt 2160 atagattttt ataaacaatc tatatctaaa catccggaat ggaaagattt tgggtttcgt 2220 ttttctgata cgcaacgtta taattctata gatgaatttt atcgtgaagt ggaaaatcaa 2280 gggtataaac ttacgtttga aaatatatct gaatcttata tagattctgt ggtgaatcaa 2340 gggaaacttt atctttttca aatatataat aaagattttt ctgcgtattc taaagggcgt 2400 ccgaatcttc atacgcttta ttggaaagcg ctttttgatg aacgtaatct tcaagatgtg 2460 gtgtataaac ttaatgggga agcggaactt ttttatcgta aacaatctat accgaaaaaa 2520 ataacgcatc cggcgaaaga agcgatagcg aataaaaata aagataatcc gaaaaaagaa 2580 tctgtgtttg aatatgatct tataaaagat aaacgtttta cggaagataa attttttttt 2640 cattgtccga taacgataaa ttttaaatct tctggggcga ataaatttaa tgatgaaata 2700 aatcttcttc ttaaagaaaa agcgaatgat gtgcatatac tttctatagc gcgtggggaa 2760 cgtcatcttg cgtattatac gcttgtggat gggaaaggga atataataaa acaagatacg 2820 tttaatataa tagggaatga tcgtatgaaa acgaattatc atgataaact tgcggcgata 2880 gaaaaagatc gtgattctgc gcgtaaagat tggaaaaaaa taaataatat aaaagaaatg 2940 aaagaagggt atctttctca agtggtgcat gaaatagcga aacttgtgat agaatataat 3000 gcgatagtgg tgtttgaaga tttgaatttt gggtttaaac gtgggcgttt taaagtggaa 3060 aaacaagtgt atcaaaaact tgaaaaaatg cttatagaaa aacttaatta tcttgtgttt 3120 aaagataatg aatttgataa aacggggggg gtgcttcgtg cgtatcaact tacggcgccg 3180 tttgaaacgt ttaaaaaaat ggggaaacaa acggggataa tatattatgt gccggcgggg 3240 tttacgtcta aaatatgtcc ggtgacgggg tttgtgaatc aactttatcc gaaatatgaa 3300 tctgtgtcta aatctcaaga atttttttct aaatttgata aaatatgtta taatcttgat 3360 aaagggtatt ttgaattttc ttttgattat aaaaattttg gggataaagc ggcgaaaggg 3420 aaatggacga tagcgtcttt tgggtctcgt cttataaatt ttcgtaattc tgataaaaat 3480 cataattggg atacgcgtga agtgtatccg acgaaagaac ttgaaaaact tcttaaagat 3540 tattctatag aatatgggca tggggaatgt ataaaagcgg cgatatgtgg ggaatctgat 3600 aaaaaatttt ttgcgaaact tacgtctgtg cttaatacga tacttcaaat gcgtaattct 3660 aaaacgggga cggaacttga ttatcttata tctccggtgg cggatgtgaa tgggaatttt 3720 tttgattctc gtcaagcgcc gaaaaatatg ccgcaagatg cggatgcgaa tggggcgtat 3780 catatagggc ttaaagggct tatgcttctt gggcgtataa aaaataatca agaagggaaa 3840 aaacttaatc ttgtgataaa aaatgaagaa tattttgaat ttgtgcaaaa tcgtaataat 3900 tag 3903

Claims (16)

DNA cleavage activity is inactivated (nuclease-deactivated) Cas12a protein (dCas12a); and
A composition for suppressing gene expression comprising a crRNA (CRISPR RNA) comprising a repetitive nucleotide sequence (direct repeat, DR) and a protospacer. The composition for inhibiting gene expression according to claim 1, wherein the repetitive nucleotide sequence is 10 nt to 46 nt in length. The composition for inhibiting gene expression according to claim 1, which is a protospacer in the T strand direction of the template DNA. The composition for inhibiting gene expression according to claim 1, for inhibiting gene expression in cyanobacteria. The composition for inhibiting gene expression according to claim 1, wherein in the crRNA, two or more different repetitive nucleotide sequences and protospacers are sequentially repeated. The composition for inhibiting gene expression according to claim 5, which can simultaneously inhibit multiple gene expression. a nucleic acid sequence encoding a Cas12a protein (dCas12a) having inactivated DNA cleavage activity;
a nucleic acid sequence encoding a crRNA including a repetitive nucleotide sequence and a protospacer; and
A recombinant vector for suppressing gene expression comprising a promoter operably linked to the nucleic acid sequence. The recombinant vector for inhibiting gene expression according to claim 7, wherein in the crRNA, two or more different repetitive nucleotide sequences and protospacers are sequentially repeated. The recombinant vector for inhibiting gene expression according to claim 8, which is capable of simultaneously inhibiting expression of multiple genes. The recombinant vector for suppressing gene expression for cyanobacteria according to claim 7 . The recombinant vector for inhibiting gene expression according to claim 7, wherein the repetitive nucleotide sequence is 10 nt to 46 nt in length. The recombinant vector for suppressing gene expression according to claim 7, which is a protospacer in the T strand direction of the template DNA. A kit for suppressing gene expression, comprising the composition of claim 1 or the vector of claim 7. 1) constructing the vector of claim 7 including a protospacer complementary to the target gene; and
2) A method of inhibiting the expression of a target gene, comprising transforming the vector into a cell or organism. 15. The method of claim 14, wherein the vector comprises a crRNA in which two or more sequentially repeated protospacers complementary to a repetitive nucleotide sequence and one or more different target genes are repeated. 16. The method of claim 15, which inhibits expression of one or more different target genes.

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