CRISPR-Cas9-based linearization of plasmid DNA templates

Abstract

The present invention relates to a method for producing a linear double-stranded DNA molecule using a guide DNA and a Cas nuclease, wherein the linear DNA molecule has blunt ends and the linear DNA molecule has a poly-T sequence at the 5' end of the template strand.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a linear double-stranded DNA molecule, wherein the linear DNA molecule has a blunt end, and the linear DNA molecule has a poly-T sequence at the 5' end of the template strand. (1) On the above template chain in the direction from 3' to 5': (i) A template sequence operably linked to an RNA polymerase promoter, (ii) PolyT sequence, (iii) Nucleotide sequences or nucleotide analogues described by N1N2N3 (wherein N1~N3 are each independently G, T, A or C), and (iv) Protospacer adjacent motif (PAM), The steps include preparing a double-stranded circular DNA molecule comprising and (2) A step of preparing a guide RNA comprising the 5' end of the polyT sequence (ii) and a region complementary to the nucleotide sequence (iii) and a region that interacts with Cas nuclease, (3) Steps to prepare type II Cas nuclease, (4) The step of contacting the double-stranded circular DNA molecule with the guide RNA and the Cas nuclease, The present invention relates to a method comprising the above. [Background technology]

[0002] Ribonucleic acid (RNA) plays a crucial role in many cellular processes. According to the central dogma of biology, they mediate the transfer of DNA to proteins and play vital roles in transcription and translation in the form of messenger RNA (mRNA) and transfer RNA (tRNA). However, many other processes, particularly at a regulatory level, namely the fine-tuning of biochemical reactions inside and outside cells, are also significantly controlled by RNA. Therefore, it is not surprising that RNA is at the center of much research work to better understand these processes. In addition to foundational work toward a general understanding of RNA, what has been discovered particularly in recent years is how useful RNA is for therapeutic applications, for example, for vaccines against SARS-CoV-2. For such applications, it is important that appropriate methods are available that enable the production of sufficient quantities of mRNA. The above methods use natural molecules, such as RNA polymerase, which produces RNA. Synthetic production of RNA by DNA-dependent RNA polymerase is a method routinely used for research and industrial applications. The above method requires a DNA template, RNA polymerase (e.g., T7 RNA polymerase), nucleotides such as ATP, CTP, GTP, and UTP, and appropriate buffer conditions. The polymerase then synthesizes the corresponding complementary RNA according to the DNA template. The RNA produced can be of different types: mRNA, self-replicating RNA (saRNA), long non-coding RNA (lncRNA), guide RNA (gRNA), small interfering RNA (siRNA), microRNA (miRNA), circular RNA (circRNA), and others. For relatively small RNAs, chemical synthesis can also be used industrially, in particular. However, chemical synthesis is not economical for relatively long RNAs exceeding 100 nucleotides, such as mRNA, and consequently, relatively long RNAs are usually produced by enzymatic in vitro transcription.

[0003] In large-scale production, plasmid DNA is used as a DNA template for in vitro transcription because, unlike DNA templates produced by PCR, plasmid DNA can be produced in the required quantity by fermentation. The self-contained circular nature of plasmids theoretically leads to infinitely long RNA transcription in RNA synthesis; in reality, it always results in at least some unnecessarily heterogeneous and elongated RNA. This elongation negatively impacts the efficiency of the transcription process, thereby increasing production costs. Two possible approaches to avoid unnecessarily elongated RNA transcription are described in the literature.

[0004] One solution is the classical use of type II restriction endonucleases. These enzymes recognize specific DNA sequences and, generally, perform specific cleavage in or near the DNA double strand within the aforementioned sequence. Such cleavage in plasmid DNA allows for so-called in vitro run-off transcription, where RNA polymerase can rejoin the "missing" promoter at the double-strand break and initiate a new synthetic round. Classical type II restriction enzymes are typically used for such cleavage in DNA templates. Restriction enzymes of subgroup IIP cleave DNA at a predetermined location within their recognition sequence. Examples of IIP restriction enzymes are EcoRI and XbaI. Alternatively, enzymes of subgroup IIS cleave outside the recognition sequence can also be used. Using the two classifications of type II restriction enzymes (IIP and IIS), linear DNA fragments can be constructed from circular plasmid DNA, with the former being used for more efficient RNA synthesis. Note that the DNA template should not contain any further recognition sequences for any specific restriction enzyme used. Therefore, the template sequence is subject to constraints, and its design is more complex. This can affect individual base pairs, but it can also affect entire codons in the DNA template or domains of the target protein, such as motifs or structures. In addition, many type II restriction enzymes, particularly type IIS enzymes, process DNA with overhangs, generating sticky ends at the so-called 5' or 3' ends. 3' overhangs are particularly undesirable because they can result in longer transcripts than originally intended due to RNA polymerase confusion. To prevent these undesirable transcripts, the overhangs must be degraded after the restriction step. Therefore, there are clearly more steps involved in planning and constructing DNA templates, increasing the complexity of the development required.

[0005] A second solution to avoid excessively long RNA transcripts is the use of Rho-dependent termination signals. In contrast to the use of restriction endonucleases, there is no linearization of the DNA template in this case. The Rho-dependent terminator is typically a secondary structure within the synthesized target RNA that prevents the generation of excessively long RNA transcripts by terminating synthesis. After synthesis has terminated, the polymerase can recombine with the promoter and initiate a new synthesis round, similar to run-off transcription. High transcription rates are maintained, but undesirable sequence regions, such as termination sequences, are also added. To remove these undesirable elements, RNA cleavage enzymes, such as RNases, as well as catalytically active nucleic acids, can be used. An example of this is DNAzyme, but also so-called ribozyme. Ribozyme is a structured RNA molecule that cleaves RNA at a predetermined site in trans (outside-derived) or in cis (sequence-derived). This generally involves cleaving specific RNA structures and divalent metal ions, usually Mg 2+ This requires Mg. This results in a nucleophilic attack on the target sequence, which in turn cleaves and, in the case of RNA synthesis, can remove excess elements. T7 RNA polymerase is Mg 2+ Since it is also ion-dependent, the ribozyme activity in the synthesis reaction is Mg 2+ Competition for ions affects the efficiency of in vitro transcription. Therefore, further optimization of reaction conditions is necessary, which can lead to decreased efficiency. When designing a suitable sequence with Cis ribozymes, it must be ensured that at least one ribozyme following a stop sequence is incorporated into the target RNA. Consequently, the entire process of manufacturing DNA templates for mRNA production becomes more expensive and therefore less economical.

[0006] For the enzymatic production of functionally mature mRNA and saRNA that should be active in mammalian cells, it is absolutely necessary for RNA to have a poly(A) tail. This poly(A) tail increases RNA stability and also plays a crucial role during translation in eukaryotic target cells. In target cells, the poly(A) tail is recognized and bound by poly(A)-binding proteins. These bound proteins trigger an interaction between translational proteins and the 3' end of the mRNA at the 5' end. This complex interaction increases not only RNA stability but also translation efficiency. Therefore, the poly(A) tail plays a vital role within mature mRNA. To provide mRNA or saRNA that is as natural as possible, the poly(A) tail should be as long as possible (>100 A nucleotides) and ideally uniform (without other bases; C, G, or U). The poly(A) tail at the 3' end of the RNA is then called the unbound poly(A) tail.

[0007] Currently, there are two conventional methods for producing RNA molecules with uniform poly(A) tails. Firstly, after synthesizing the target RNA sequence, poly(A) tails can be produced using poly(A) polymerase. In the enzymatic step, the produced RNA is first purified, and then the poly(A) tails are synthesized by adding ATP as both poly(A) polymerase and substrate. The length of the reaction time determines the average length of the poly(A) tails. While this enzymatic synthesis generally works very well, it does have a few drawbacks. For example, it is not possible to produce poly(A) tails of clearly defined lengths with this technique; what is produced is a product containing heterogeneous RNA molecules with a normal distribution of these lengths. Therefore, the industrial use of RNA molecules produced in this way requires further processing steps for RNA enrichment or purification, which reduces yield and increases production costs.

[0008] In contrast, this is a method in which the poly-A tail is directly encoded by a DNA template that serves as a template for RNA production. For this purpose, with respect to polyadenylation, this must be ensured if the template DNA is designed so that the double-stranded DNA contains the required poly-T motif on the template strand. This method has the advantage of producing many more defined products with poly-A tails of uniform length in a single reaction step.

[0009] When PCR products are used as templates for the synthesis of RNA molecules with a uniform and uncoated poly(A) tail, the poly(A) tail can be directly encoded in the primers used for PCR. To produce DNA templates by PCR, reverse primers containing a sequence of up to 120 or more adenines, in addition to the complementary sequence at the 3' end, can be used. PCR adds this tail to the coding DNA, making it available for in vitro transcription. Since PCR reactions can technically only be performed in small volumes of a few hundred microliters or less, large-scale RNA production processes require hundreds of PCR reactions. Upscaling PCR reactions by so-called scale-out, where multiple reactions are performed in parallel, is currently not economical due to the high workload and cost associated with industrial-scale operations.

[0010] When plasmid DNA is used as a template for in vitro transcription of RNA, this requires plasmid fermentation, pDNA purification, and subsequent linearization of the pDNA. After linearization, the linearized pDNA is purified to obtain the purest possible DNA template for in vitro transcription, thereby increasing the complexity and cost of the method. As already noted, a drawback of using IIP restriction enzymes is that these enzymes do not yield a free, uncovered poly(A) tail, as the recognition sequences of IIP restriction enzymes always contain little to no other nucleotides such as C, G, or T. In this regard, this improved method is provided by using IIS-type enzymes, as they can cleave outside these recognition sequences, and thus the desired uncovered poly(A) tail can be produced. However, the use of IIS-type enzymes usually results in 3' and 5' overhangs that need to be degraded first, as mentioned above.

[0011] In addition to the use of restriction enzymes, ribozymes can also be used for linearization of template DNA. These can also be designed to produce uniform and free poly(A) tails. It is preferable to design the ribozymes to directly cleave at the ends of the poly(A) sequence. The drawback of ribozymes is that, in the case of trans ribozymes, additional RNA must be produced, and in the case of cis ribozymes, obviously longer RNA must be produced. This approach increases the complexity of production and, therefore, the cost.

[0012] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-related (Cas) nucleases have recently been identified as components of the immune system. Because Cas nucleases can precisely cleave DNA elements at specific sites, the CRISPR-Cas system has been developed as an effective gene editing tool, primarily for applications in the field of genome editing, and has revolutionized molecular biology and microbiology in this field (Hille et al., Cell 172, Issue 6, 1239-1259, 2018; Plagens et al. FEMS Microbiology Reviews, 39(3):442-63, 2015; Richter et al. Int J Mol Sci. 2013 Jul; 14(7): 14518-14531).

[0013] When bacterial cells are infected with foreign nucleic acids, such as phage DNA, transposons, pDNA, or RNA, the bacteria react with an immune response to eliminate the foreign DNA or RNA. It is worth noting that CRISPR-Cas immunity is an adaptive immunity, meaning that this type of immune response is learned by the bacterial cell. During infection, the immune system uses short DNA fragments of the foreign nucleic acid, which can consequently induce a faster response later in similar events of infection. This DNA fragment is incorporated into a gene locus for the CRISPR-Cas system in the CRISPR array, also called a spacer. This spacer sequence later becomes part of the guide RNA (gRNA) or CRISPR RNA (crRNA), and thus contributes to sequence specificity. As part of this bacterial immune defense, the Cas nuclease is directed by the crRNA towards a specific target sequence on the foreign nucleic acid. Sequence recognition requires, firstly, complementarity of the crRNA with the foreign target DNA, and secondly, a short signal sequence immediately adjacent to the target DNA called a protospacer-adjacent motif (PAM). Depending on the CRISPR-Cas system, a PAM sequence consists of a specific number of nucleotides and is an initiation recognition sequence for Cas nucleases on the DNA to be cleaved. For example, the Cas nuclease Cas9 (SpCas9) from Streptococcus pyogenes recognizes the PAM sequence 5'-NGG-3'. If there is no PAM sequence immediately adjacent to the target sequence, the foreign DNA will not be cleaved. After recognition by the Cas nuclease and crRNA ribonucleoprotein complex, the foreign target sequence is specifically cleaved, and the foreign DNA can then be further degraded by cellular nucleases. Only the appropriate combination of PAM sequence and target sequence in appropriate spatial proximity results in Cas nuclease cleavage activity. Since specific combinations of PAM sequence and target sequence generally do not exist in bacterial genomes, autoimmune reactions, i.e., cleavage of endogenous DNA, are prevented.

[0014] In particular, different CRISPR-Cas systems and CRISPR complexes, unlike those found in nature, require a certain number of proteins to bind and cleave at precise sites in the target sequence. A particularly well-understood system is the Type II-A system, which uses the Cas9 protein as an effector nuclease for interference with the target DNA. In addition to the title CRISPR array, which encodes various crRNAs, Type II systems include trans-activating RNA (tracrRNA), adaptive proteins Cas1, Cas2, and Cas4, and the effector nuclease Cas9. The tracrRNA generates a hybrid with the crRNA encoded by the CRISPR array. This hybrid then allows Cas9 to specifically bind. The crRNA portion of the hybrid construct directs Cas9 towards the target DNA. Because the two-component system of crRNA and tracrRNA is relatively cumbersome for molecular biology, crRNA and tracrRNA are often linked together by a linker. This resulting RNA is called sgRNA (single-stranded guide RNA). [Overview of the project]

[0015] An object of the present invention is to provide a simplified or improved linearization method for circular DNA molecules that can be used as DNA templates for enzymatic RNA synthesis. To achieve this object, the linearized DNA molecule should have blunt ends and a poly-T sequence at the 5' end of the template strand. In particular, the above method provides a linearized DNA molecule that enables the synthesis of RNA having a uniform and uncovered poly-A tail. A further object of the present invention is to provide a method that requires fewer steps than methods disclosed in the prior art. A further object of the present invention is to provide a method for obtaining linear DNA molecules with improved uniformity with respect to the length of the poly-A tail. A further object of the present invention is to provide a method that can be carried out more effectively or more cost-effectively than methods described in the prior art.

[0016] The above object is a method for producing a linear double-stranded DNA molecule, wherein the linear DNA molecule has blunt ends, and the linear DNA molecule has a poly-T sequence at the 5' end of the template strand: (1) On the template strand in the 3' to 5' direction (i) A template sequence operably linked to an RNA polymerase promoter, (ii) A poly-T sequence, (iii) A nucleotide sequence described by N1N2N3 (where N1 to N3 are each independently G, T, A or C) or a nucleotide analog thereof, and (iv) A protospacer adjacent motif (PAM), preparing a double-stranded circular DNA molecule comprising: (2) preparing a guide RNA comprising a region complementary to the 5' end of the poly-T sequence (ii) and the nucleotide sequence (iii) and a region that interacts with the Cas nuclease; (3) preparing a type II Cas nuclease; (4) contacting the double-stranded circular DNA molecule with the guide RNA and the Cas nuclease; is achieved by a method comprising:

[0017] In particular, the application of the CRISPR-Cas system in the field of linearization of plasmid DNA as a template for in vitro transcription of RNA is not yet known. The combination of guide RNA, Cas nuclease, such as Cas9, and double-stranded circular DNA molecule allows the CRISPR-Cas system to be used for efficient and uniform linearization of plasmid DNA.

[0018] Thanks to the possibility of flexible design of the guide RNA, the linearization method can be used flexibly for all double-stranded circular DNA molecules, enabling specific cleavage at a pre-selected site of the DNA.

[0019] Thanks to the flexible design possibilities of PAM, the linearization method can be flexibly implemented using different Cas nucleases.

[0020] Upon cleavage of the double strand in the target molecule, type II Cas nuclease, such as Cas9, generates a so-called "blunt" DNA end, that is, a DNA end without a 3' overhang or a 5' overhang. Such a DNA template with a blunt end is particularly highly suitable for RNA synthesis. In mRNA synthesis, it is desirable that the obtained mRNA has a uniform and uncovered polyA tail of a clearly defined length. The more uniform the polyA tail of the mature mRNA, that is, the fewer non-adenine bases, the easier and more effective the polyA-binding protein is. Next, the binding of this protein is important for the effective translation of the mature mRNA in the target cell. Therefore, the adverse effects of mRNA having no direct adenine residues or ends with different bases, which occur when using many type II restriction enzymes for example, can be avoided. Therefore, the method according to the present invention enables the production of linear DNA templates that are particularly highly suitable for the production of natural, mature RNA, such as mRNA having advantages for therapeutic applications, such as improved stability, a relatively long half-life or a relatively high translation efficiency.

[0021] The method according to the present invention provides a high degree of freedom in sequence design and absolute sequence dependence, that is, there are no restrictions on nucleotides as well as on sequence and structural motifs.

[0022] The CRISPR-Cas-based method according to the present invention depends on methylation and other DNA modifications when it is a case of using many restriction enzymes.

[0023] According to the present invention, linearization of double-stranded circular DNA molecules, i.e., DNA templates, can occur during plasmid DNA replication, i.e., for example, within bacterial-producing cells or before cell disruption. This has the advantage of reducing the number of processing steps. Therefore, at the end of pDNA fermentation, linearization by a Cas nuclease, such as Cas9, can already occur within the cell. After completion of fermentation and intracellular linearization, the linear DNA can be purified by conventional methods; there is no need to separate the double-stranded circular DNA molecules. Subsequently, the linear DNA template can be used directly in in vitro transcription for the production of RNA, for example, mRNA having an uncoated poly(A) tail.

[0024] The linear double-stranded DNA molecule produced by the present invention has a blunt end and a poly-T sequence at the 5' end of the template strand. Such a linear DNA molecule is particularly well-suited as a DNA template for in vitro transcription. In vitro transcription is an enzyme-catalyzed process in which RNA is produced in vitro, and this involves the use of a DNA-dependent RNA polymerase to synthesize RNA complementary to the DNA template. Synthesis requires ribonucleotides (NTPs), buffer systems such as TRIS-HCl, magnesium ions such as MgCl2, and optionally, RNase inhibitors, inorganic pyrophosphatases, and spermidine. Monomeric RNA polymerases, which are generally bacteriophage enzymes, are commonly used. Examples of the most commonly used polymerases are T7 RNA polymerase (UniProt: P00573) or T3 RNA polymerase (UniProt: Q778M8). Specific Description of the Invention

[0025] definition Unless otherwise specified, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in the field of the present invention. Any technical feature referred to in the following definitions may be applied to any embodiment of the present invention.

[0026] The terms “nucleic acid sequence,” “nucleotide sequence,” “DNA sequence,” and “RNA sequence” are known to those skilled in the art and refer to individual and specific sequences of nucleotides.

[0027] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to a sequence of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include single-stranded and double-stranded DNA or RNA, genomic DNA, cDNA, mRNA, saRNA, gRNA, siRNA, miRNA, or circRNA, which may include purine and pyrimidine bases, nucleotide analogs, or other natural, chemically or biochemically modified, unnatural, or derivatized nucleotide bases.

[0028] A nucleotide analog represents a chemical compound that is structurally and functionally similar to a nucleotide; that is, a nucleotide analog can be recognized as a substrate by polymerase. In this specification, a nucleotide analog is understood to mean particularly natural and unnatural variants of the natural nucleotides adenosine, cytosine, thymidine, guanosine, and uridine, such as chemically derivatized nucleotides having unnatural functional groups that are added to or removed from natural nucleotides or that substitute for natural functional groups of nucleotides. Examples of nucleotide analogs include N1-methylpseudridine, 5-methoxyuridine, 5-methylcytidine, pseudouridine, N4-acetylcytidine, and N6-methyladenine.

[0029] As used herein, a poly-T sequence shall be understood to mean a sequence of thymine nucleotides in which other nucleotides, namely adenine, guanine, or cytosine, are preferably absent.

[0030] As used herein, a polyA sequence shall be understood to mean a sequence of adenine nucleotides in which other nucleotides, namely thymine, guanine, or cytosine, are preferably absent.

[0031] A polyT or polyA sequence may be divided into two or more distinct segments, each having at least about 40 thymine or adenine nucleotides. Each individual segment may be interspersed with a sequence of about 10-20 nucleotides in length that is not a polyT or polyA sequence, and may also include G, C, and U. Such segments can provide improved stability to the polyT or polyA sequence.

[0032] It should be understood herein that an uncovered polyA tail of RNA means a terminal polyA sequence in which no nucleotides other than adenine follow the adenine nucleotide sequence at the 3' end of the RNA, i.e., no cytosine, uracil, or guanine follows.

[0033] A poly(A) tail of RNA from in vitro transcription is said to be homogeneous in this specification if, in addition to the uncovered 3' end, it has a narrow size distribution around a given target length. Preferably, the mean deviation from the target length is 10% or less, more preferably 5% or less, and particularly preferably less than 5% based on the number of nucleotides. A split poly(A) tail is considered homogeneous if, all segments combined, the whole has the above mean deviation from the overall target length of the segments. Splitting can result in stabilization of the entire poly(A) tail. In the case of a split poly(A) tail of RNA, it is advantageous that the 3' segment of the poly(A) tail is uncovered to ensure high translation efficiency.

[0034] The template strand of double-stranded DNA is understood to be the DNA strand that is read by RNA polymerase during transcription to synthesize RNA.

[0035] A template sequence, template DNA, or DNA template is understood to mean a DNA sequence used for in vitro transcription. The sequence functions as a template for DNA-dependent RNA polymerase to synthesize RNA according to the sequence.

[0036] The template sequence may consist of nucleic acids encoding antigens, such as tumor antigens, viral antigens, or bacterial antigens, therapeutic proteins, or other proteins such as growth factors or transcription factors.

[0037] As used herein, a promoter sequence or promoter is a DNA regulatory region that can bind to RNA polymerase and induce transcription of downstream (3' direction) coding or non-coding sequences. A suitable promoter may originate from any organism, including prokaryotes and eukaryotes.

[0038] When used herein, operably linked to a promoter means that the promoter brings about or regulates the transcription of DNA containing the coding of a gene, for example, a gene whose coding is defined by a template sequence, or a gene encoding a guide RNA, or a gene encoding a type II Cas nuclease. The promoter may be a native promoter, i.e., a promoter present in the cell into which the vector has been introduced. In one embodiment, the promoter is an inducible promoter, i.e., the promoter is regulated to enable the inducible transcription of a gene, such as a gene encoding a guide RNA or a gene encoding a type II Cas nuclease.

[0039] The promoter may be a constitutively active promoter, i.e., a promoter in a constitutively active state, or the promoter may be an induced promoter, i.e., a promoter whose state is controlled by an external stimulus or which moves from an inactive state to an active state. The stimulus or inducer causes the expression of the target gene, i.e., activates it. This method is preferably used when the expression or overexpression of the target gene has adverse effects on the organism or process being produced. Such external stimuli may be, for example, a specific temperature, a compound, or a protein.

[0040] Examples of induced promoters include the IPTG-induced Lac promoter (Jacob and Monod, Volume 3, Issue 3, Journal of Molecular Biology, June 1961, Pages 318-356, 1961), the tetracycline-regulated promoter (Gossen et al. 1995 Science 268 (5218): 1766-9), the rhamnose-induced promoter (Kelly et al., ACS Synth. Biol.; 2016, 5 Pages 1136-1145), and the arabinose-induced arabinose promoter (Guzman et al., J Bacteriol. 1995, Jul;177(14):4121-30).

[0041] The term “protospacer adjacency motif” or PAM refers to a signal sequence of approximately 2–8 base pairs that is a specific recognition site for a Cas nuclease or multiprotein Cas effector complex (e.g., CASCADE or CMR) on the DNA being cleaved. If a PAM signal sequence is not present, the foreign DNA will not be cleaved. The length and base sequence of the PAM sequence vary depending on the CRISPR-Cas system and the Cas nuclease, as well as on the relevant organisms in which the Cas nuclease naturally occurs. For example, the PAM sequence associated with the Cas9 nuclease (SpCas9) of Streptococcus pyogenes is 5'-NGG-3', where “N” is any nucleotide, i.e., a nucleotide that is A, T, G, or C followed by two guanines. Further chemical species and related PAM sequences are known to those skilled in the art and are described in the prior art (Fonfara et al. Nucleic Acids Research, 2014, Vol. 42, No. 4 2577-2590).

[0042] Guide RNA (gRNA) is a specific RNA sequence to which Cas nucleases can bind and direct the Cas nucleases to template DNA. The guide RNA specifically hybridizes with a specific target site on the template DNA, allowing the Cas nucleases to cleave the template DNA. The guide RNA may be a two-component guide RNA, or a hybrid of crRNA and tracrRNA. Alternatively, the guide RNA may be individual sgRNAs (single-stranded guide RNAs) in which essential regions of crRNA and tracrRNA bind to each other to produce a continuous chimeric RNA. The sgRNAs can also mimic the hairpin structure of a crRNA::tracrRNA double strand, allowing them to bind to Cas nucleases and direct them to target sequences on the template DNA. The guide RNA is designed based on sequence information about the template DNA to be cleaved.

[0043] As used herein, complementary or complementary relationships refer to a relationship between two DNA or RNA nucleic acid regions that match each other and form hydrogen bonds with each other, i.e., hybridize with each other based on these nucleic acid bases, as described by Watson and Crick. Known in the prior art are, for example, standard Watson-Crick base pairs: adenine (A) pair with thymidine (T), adenine (A) pair with uracil (U), and guanidine (G) pair with cytosine (C).

[0044] Uniform genes or sequences are understood herein to mean that the DNA sequences or sections of DNA of the above genes are at least 70% identical, preferably at least 80% identical, preferably at least 90% identical, and particularly preferably at least 95% identical. The degree of DNA identity is determined by the "nucleotide blast" program, which can be found at http: / / blast.ncbi.nlm.nih.gov / and is based on the blastn algorithm. The algorithm parameters used to align two or more nucleotide sequences were default parameters. The default general parameters are: Max target sequences=100; Short queries=“Automatically adjust parameters for short input sequences”; Expect Threshold=10; Word size=28; Automatically adjust parameters for short input sequences=0. The corresponding default scoring parameters are: Match / Mismatch Scores=1,-2; Gap Costs=Linear.

[0045] A uniform amino acid sequence is understood to be at least 70%, preferably at least 80%, preferably at least 90%, and particularly preferably at least 95% identical, where each change in a uniform sequence is the insertion, addition, deletion, and substitution of one or more amino acids. Protein sequences are compared using the "protein blast" program at http: / / blast.ncbi.nlm.nih.gov / . This program uses the blastp algorithm. The algorithm parameters used to align two or more protein sequences were the default parameters. The default general parameters are: Max target sequences=100; Short queries=“Automatically adjust parameters for short input sequences”; Expect Threshold=10; Word size=3; Automatically adjust parameters for short input sequences=0. The default scoring parameters are: Matrix=BLOSUM62; Gap Costs=Existence:11 Extension:1; Compositional adjustments=Conditional compositional score template adjustment.

[0046] Detailed description of the present invention In one embodiment, the present invention relates to a method for producing a linear double-stranded DNA molecule, wherein the linear DNA molecule has a blunt end, and the linear DNA molecule has a poly-T sequence at the 5' end of the template strand. (1) On the above template chain in the direction from 3' to 5': (i) A template sequence operably linked to an RNA polymerase promoter, (ii) PolyT sequence, (iii) Nucleotide sequences or nucleotide analogues described by N1N2N3 (wherein N1~N3 are each independently G, T, A or C), and (iv) Protospacer adjacent motif (PAM), The steps include preparing a double-stranded circular DNA molecule containing and (2) A step of preparing a guide RNA comprising the 5' end of the polyT sequence (ii) and a region complementary to the nucleotide sequence (iii) and a region that interacts with Cas nuclease, (3) Steps to prepare type II Cas nuclease, (4) The step of contacting the double-stranded circular DNA molecule with the guide RNA and the Cas nuclease, The present invention provides a method comprising the following:

[0047] Provided for the method according to the present invention is a specially designed double-stranded circular DNA molecule. The circular DNA molecule comprises an RNA polymerase promoter described by N1N2N3, a poly-T sequence, and a template sequence operably linked to a nucleotide sequence on a template strand in the 3' to 5' direction, where N1 to N3 are each independently G, T, A or C or a nucleotide analog thereof, and a protospacer adjacent motif (PAM).

[0048] The RNA polymerase promoter is preferably a recognition sequence for DNA-dependent RNA polymerase, such as the T7 RNA polymerase promoter, the SP6 RNA polymerase promoter, or the T3 RNA polymerase promoter.

[0049] The template sequence may include, for example, nucleic acids encoding an antigen, such as a tumor antigen, viral antigen, or bacterial antigen. In another example, the template sequence may include nucleic acids encoding a therapeutic protein or several other proteins, such as a growth factor or transcription factor.

[0050] The polyT sequence on the template strand of a double-stranded circular DNA molecule consists of, for example, about 40 to about 250 T nucleotides. Preferably, the polyT sequence consists of about 100 to about 140 T nucleotides, and more preferably about 120 T nucleotides.

[0051] In the nucleotide sequence described by N1N2N3, N1 to N3 are each independently G, T, A, or C or a nucleotide analog. Preferably, N1 to N3 are each independently G, T, A, or C. In a particularly preferred embodiment, N1 to N3 are each independently G, A, or C.

[0052] In addition, the protospacer adjacent motif (PAM) is located on the template strand of the double-stranded circular DNA molecule. Preferably, the PAM is located immediately after the nucleotide sequence N1N2N3.

[0053] The PAM may be any selected PAM depending on the specific Cas nuclease used in the method according to the present invention. For example, the PAM comprises a sequence recognized by a type II Cas nuclease. For example, the PAM is selected from NGG, NGGNG, NNAAAAW, NG, NNNNACA, NNNNGATT, GNNNCNNA, and NNAGNN, in all cases, N is independently selected from G, C, A, and T, and the PAM sequence is shown in the 5' to 3' direction. In a preferred embodiment, the PAM comprises the sequence 5'-NGG-3', and N is independently selected from G, C, A, and T. In another preferred embodiment, the PAM has the sequence 5'-NGG-3', and N is independently selected from G, C, A, and T. In a further preferred embodiment, the PAM is recognized by a Cas9 nuclease derived from Streptococcus.

[0054] The target site for cleavage of the circular double-stranded DNA is preferably three nucleotides from the PAM in the 5' direction.

[0055] A guide RNA for the method according to the present invention is designed to have a sequence at the 5' end that is complementary to the template strand of a double-stranded circular DNA molecule. This complementary region consists of, for example, about 20 nucleotides complementary to about 20 consecutive nucleotides of the template strand.

[0056] The guide RNA comprises a region complementary to the 5' end of the polyT sequence (ii) and the nucleotide sequence (iii). Preferably, the region of the guide RNA complementary to the 5' end of the polyT sequence (ii) and the nucleotide sequence (iii) is sequence 5'-G(A) n- It has N4N5N6-3'. In this case, n is a number between 17 and 23, preferably a number between 17 and 20, and more preferably n is equal to 17.

[0057] N4 to N6 are each independently G, U, A, C or a nucleotide analog, preferably N4 to N6 are each independently G, U, C or a nucleotide analog, and particularly preferably N4 to N6 are each independently G, U or C.

[0058] The complementary sequence within the guide RNA has, for example, the sequence 5'-GAAAAAAAAAAAAAAAAANNN-3', and in all cases, N is independently any of the nucleotides. Preferably, the above method is characterized in that the guide RNA is Sequence ID No. 1.

[0059] The complementarity of the region in the guide RNA to the 5' end of the poly-T sequence (ii) and the nucleotide sequence (iii) is at least 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Preferably, the complementarity is 98%, 99%, or 100%, and particularly preferably 100%.

[0060] The nucleotides N4N5N6 of the guide RNA are complementary to the nucleotides N1N2N3 from nucleotide sequence (iii) of the template strand. This means that N4 is complementary to N1, N5 is complementary to N2, and N6 is complementary to N3. Preferably, the complementarity between nucleotides N4N5N6 and nucleotides N1N2N3 is 100%. Preferred complementary base pairs include, for example, G and C, as well as A and T or A and U. Complementary sequences are, for example, CAG for N1N2N3 and GUC for N4N5N6.

[0061] Furthermore, the guide RNA includes a region that interacts with Cas nucleases. Preferably, the guide RNA includes a region that interacts with type II Cas nucleases.

[0062] Furthermore, the method according to the present invention provides a type II Cas nuclease. Any type II Cas nuclease may be used. For example, Streptococcus pyogenes (SpCas9;UniProt Q99ZW2), Streptococcus mutans (SmCas9;UniProt Q8DTE3), Streptococcus thermophilus (StCas9;UniProt G3ECR1), Streptococcus aureus (SaCas9;UniProt J7RUA5), Francisella novicida (FnCas9;UniProt A0Q5Y3), Campylobacter jejuni (CjCas9;UniProt A0A698TVI8), Neisseria meningitidis We provide type II Cas nucleases derived from *Streptococcus meningitids* (MnCas9; UniProt C9X1G5) and *Pasteurella multocida* (PmCas9; AddGene#68703; https: / / www.addgene.org / 68703 / ). Preferably, we provide type II Cas nucleases derived from *Streptococcus spec.*. More preferably, we provide type II Cas nucleases derived from *Streptococcus pyogenes*. More preferably, the type II Cas nuclease used is Cas9 derived from *Streptococcus pyogenes*.

[0063] Preferably, the type II Cas nuclease used is Cas9. For example, Cas9 is selected from Streptococcus pyogenes having sequence number 2 or a sequence that is at least 70% homologous thereto, preferably at least 80% homologous, more preferably at least 90% homologous, more preferably at least 95% homologous, and particularly preferably at least 98% homologous; Streptococcus aureus having sequence number 3 or a sequence that is at least 70% homologous thereto, preferably at least 80% homologous, more preferably at least 90% homologous, more preferably at least 95% homologous, and particularly preferably at least 98% homologous; and Streptococcus thermophilus having sequence number 4 or a sequence that is at least 70% homologous thereto, preferably at least 80% homologous, more preferably at least 90% homologous, more preferably at least 95% homologous, and particularly preferably at least 98% homologous.

[0064] In a preferred embodiment, the type II Cas nuclease is Cas9 derived from Streptococcus pyogenes having SEQ ID NO: 2, or a sequence that is at least 70% homologous thereto, preferably at least 80% homologous, more preferably at least 90% homologous, more preferably at least 95% homologous, and particularly preferably at least 98% homologous.

[0065] If the Cas nuclease used in the method according to the present invention is Cas9 derived from S. pyogenes, the PAM used is preferably 5'-NGG-3'. If the Cas nuclease used is Cas9 derived from S. mutans, the PAM used is preferably 5'-NGG-3'. If the Cas nuclease used is a Cas9 variant derived from S. thermophilus, the PAM used is preferably 5'-NGGNG-3' or 5'-NNAAAAW-3'. If the Cas nuclease used is Cas9 derived from Francisella novicida, the PAM used is preferably 5'-NG-3'. If the Cas nuclease used is Cas9 derived from Campylobacter jejuni, the PAM used is preferably 5'-NNNNACA-3'. If the Cas nuclease used is Cas9 derived from Neisseria menigitidis, the PAM used is preferably 5'-NNNNGATT-3'. If the Cas nuclease used is Cas9 derived from Pasteurella multocida, the PAM used is preferably 5'-GNNNCNNA-3', and the present invention is not intended to be limited to these examples.

[0066] In a further preferred embodiment, the present invention relates to a method for producing a linear double-stranded DNA molecule, wherein the linear DNA molecule has a blunt end, and the linear DNA molecule has a poly-T sequence at the 5' end of the template strand. (1) On the above template chain in the direction from 3' to 5': (i) A template sequence operably linked to an RNA polymerase promoter, (ii) A polyT sequence consisting of approximately 120 T nucleotides, (iii) Nucleotide sequences or nucleotide analogues described by N1N2N3 (wherein N1~N3 are each independently G, T, A or C), and PAM having sequence 5'-NGG-3' (where N is independently selected from G, C, A and T), The steps include preparing a double-stranded circular DNA molecule containing and (2) A step of preparing a guide RNA comprising the 5' end of the polyT sequence (ii) and a region complementary to the nucleotide sequence (iii), and a region that interacts with a Cas nuclease, wherein the region complementary to the 5' end of the polyT sequence (ii) and the nucleotide sequence (iii) is sequence 5'-G(A) n A step having N4N5N6-3' (where n is equal to 17, and N4~N6 are each independently G, U, or C), (3) A step of preparing the above-mentioned type II Cas nuclease, wherein the above-mentioned type II Cas nuclease is Cas9 derived from Streptococcus pyogenes having sequence number 2, or a sequence that is at least 70% homologous thereto, (4) The step of contacting the double-stranded circular DNA molecule with the guide RNA and the Cas nuclease, The present invention provides a method comprising the following:

[0067] In the method according to the present invention, the guide RNA may be prepared as an RNA molecule. According to the present invention, the guide RNA may be prepared by the expression of a nucleotide sequence encoding the guide RNA. Preferably, the guide RNA is prepared by the expression of a nucleotide sequence encoding the guide RNA.

[0068] In the method according to the present invention, type II Cas nuclease may be prepared as a protein. According to the present invention, type II Cas nuclease may be prepared by expression of a nucleotide sequence encoding type II Cas nuclease. Preferably, type II Cas nuclease is prepared by expression of a nucleotide sequence encoding type II Cas nuclease. Particularly preferably, both guide RNA and Cas nuclease are prepared by expression of nucleotide sequences. The nucleic acid encoding guide RNA and / or Cas nuclease may be located on an expression plasmid. For example, the nucleic acid encoding guide RNA may be located on one expression plasmid, and the nucleic acid encoding Cas nuclease may be located on another expression plasmid. In another example, the nucleic acid encoding guide RNA and the nucleic acid encoding Cas nuclease are located on the same expression plasmid.

[0069] For example, an expression plasmid containing Cas9 and guide RNA may be prepared as follows: Using cloning, the Cas9 nuclease may first be cloned into a suitable expression plasmid, and then, for example, the tracrRNA construct and minimal CRISPR array may be cloned in the 5'-3' direction. This allows, for example, the natural formation of the tracrRNA::crRNA double-stranded guide RNA. A suitable expression vector is, for example, pWAC003 (SEQ ID NO: 5), which is a low-copy plasmid containing approximately 10 copies per cell based on the pACYC origin of replication.

[0070] According to the present invention, what happens is that a double-stranded circular DNA molecule is brought into contact with guide RNA and a CAS nuclease. The contact may be carried out, for example, in vitro. In embodiments in which the preparation of the guide RNA and / or type II Cas nuclease is carried out by the expression of a nucleotide sequence encoding the guide RNA and / or type II Cas nuclease, the contact is preferably carried out intracellularly, particularly in bacterial cells in which plasmid DNA can be increased.

[0071] Constitutive expression of Cas endonucleases, such as Cas9, can induce nonspecific DNA cleavage events at various sites in plasmid products, i.e., double-stranded circular DNA molecules, or the chromosomes of the bacteria used. Therefore, since the free ends of linear DNA are recognized by the corresponding DNase, premature treatment of double-stranded circular DNA molecules with intracellular Cas nucleases, such as Cas9, leads to the degradation of the desired linear DNA product. Furthermore, the cell as a producer may also be damaged. To mitigate this effect, it is preferable to use a regulated promoter with minimal basic expression for type II Cas nucleases, such as Cas9.

[0072] In addition, for the induced expression of Cas nucleases, such as Cas9, the possible limit of promoter depressurization, i.e., the possible limit of induced expression level, is particularly preferred. Overly high expression of Cas nucleases, such as Cas9, within a short timeframe can have adverse effects on producer cells similar to those with high basal expression over a relatively long period of time and pDNA quality. Therefore, promoters with low basal expression and moderate post-induced expression levels are particularly preferred.

[0073] The advantage of guide RNA-induced expression over constitutive expression is that its expression does not cause constant stress to the producer cell. As a result of lower stress, the cell can produce more of the actual target product, circular DNA.

[0074] The use of controllable or induced promoters allows for the production of Cas nucleases and guide RNAs that are specifically selected by the induction of expression. Controllable or induced expression of the processing device, i.e., intracellular Cas9 nucleases and guide RNAs, eliminates the need for individual steps in subsequent processes, which offers economic advantages. For example, additional linearization steps or separate purification of plasmid DNA may be omitted.

[0075] Preferably, the method according to the present invention is characterized by the inducible expression of type II Cas nuclease and / or guide RNA. This means that the nucleotide sequence encoding the guide RNA and / or the nucleotide sequence encoding the Cas nuclease are operably ligated to an inducible promoter. Preferably, both the nucleotide sequence encoding the guide RNA and the nucleotide sequence encoding the Cas nuclease are operably ligated to an inducible promoter.

[0076] In one embodiment, the nucleotide sequences encoding the guide RNA and the nucleotide sequences encoding the Cas nuclease are operably linked to an inducible promoter. When the same or a similar inducible system is used, the expression of the guide RNA and the Cas nuclease is initiated simultaneously. Firstly, this producer allows for a reduction in cellular stress, and secondly, the synchronized initiation of expression allows for a more appropriate ratio of the molecules, Cas endonuclease to guide RNA.

[0077] In another embodiment, the nucleotide sequences encoding the guide RNA and the nucleotide sequences encoding the Cas nuclease are operably linked to different inducible promoters for sequential expression, for example.

[0078] Inducible promoters are known to those skilled in the art. Suitable examples of inducible promoters are the lac, tac, trc, λPL, ara, cumate, or tet promoters or sequences derived therefrom.

[0079] Preferably, according to the present invention, the inducible promoter used is a lactose-induced promoter, in particular the promoter WAC003 having the sequence of SEQ ID NO: 6 or the promoter WAC003-LacIDel having the sequence of SEQ ID NO: 7.

[0080] In one embodiment, the nucleotide sequence encoding the guide RNA and / or the nucleotide sequence encoding the Cas nuclease are integrated into the genome of Escherichia coli (E. coli). In a preferred embodiment, the nucleotide sequence encoding the guide RNA and the nucleotide sequence encoding the Cas nuclease are integrated into the genome of Escherichia coli (E. coli). In another preferred embodiment, the nucleotide sequence encoding the Cas nuclease is integrated into the genome of Escherichia coli (E. coli), but the nucleotide sequence encoding the guide RNA is not. Methods for integrating nucleotide sequences into bacterial genomes are known to those skilled in the art (e.g., Datsenko et al. 2000, Proc. Natl. Acad. Sci. US A. 97: 6640 - 5).

[0081] In another embodiment, the present invention relates to the use of a type II Cas nuclease for linearization of a double-stranded circular DNA molecule, wherein the linear DNA molecule is arranged on the template strand in the 3' to 5' direction. (i) A template sequence operably linked to an RNA polymerase promoter, (ii) PolyT sequence, (iii) Nucleotide sequences or nucleotide analogues described by N1N2N3 (wherein N1~N3 are each independently G, T, A or C), and (iv) Protospacer adjacent motif (PAM), It provides a use that includes the following.

[0082] In a further embodiment, the present invention provides a use for producing a linear double-stranded DNA molecule of a recombinant microorganism integrated into its genome, wherein a nucleotide sequence encoding a guide RNA and / or a nucleotide sequence encoding a type II Cas nuclease is provided, the linear double-stranded DNA molecule having blunt ends and a poly-T sequence at the 5' end of the template strand.

[0083] One embodiment provides the use of recombinant microorganisms in which a nucleotide sequence encoding guide RNA and a nucleotide sequence encoding type II Cas nuclease are incorporated into the genome. Another embodiment provides the use of recombinant microorganisms in which a nucleotide sequence encoding type II Cas nuclease is incorporated into the genome for the production of linear double-stranded DNA molecules.

[0084] The recombinant microorganism may be any microorganism suitable for plasmid DNA production. Preferably, the recombinant microorganism is Escherichia coli (E. coli), more preferably strain K12 of Escherichia coli, and particularly preferably strain NEB10β of Escherichia coli.

[0085] The present invention will be described in further detail below with reference to exemplary embodiments and accompanying drawings, but will not be limited thereto. [Brief explanation of the drawing]

[0086] [Figure 1] Agarose gel containing plasmid DNA separated by gel electrophoresis and various controls: 1: Marker (GeneRuler® 1kb DNA ladder) 2: pGFP1, no incubation (negative control 1) 3: pGFP1 aqueous solution, with incubation (negative control 2) 4: pGFP1, cleavage buffer solution (negative control 3) 5: pGFP1, cleavage buffer + 1mM EDTA solution (negative control 4) 6: pGFP1, incubated with Xbal (positive control 1) 7: pGFP1, incubated with Ncol (positive control 2) 8: pGFP1, incubated with 25nM Cas9 (negative control 5) 9: pGFP1, incubated with 25nM sgRNA (negative control 6) 10-15: Test samples 1-5 (1x, 2x, 3x, 4x, 5x and 10x excess Cas9 / guide RNA relative to pDNA). [Figure 2]The image shows an agarose gel containing mRNA separated by gel electrophoresis. The mRNA was generated by in vitro transcription using different DNA templates. Cas9 represents a sample using linearized DNA with SpCas9 / guide RNA as the template DNA. IIS type represents a sample using linearized DNA with IIS restriction enzyme as the template DNA. PCR represents a sample using DNA produced by PCR as the template. pGFP and pGFP2 represent samples using circular plasmids pGFP1 and pGFP2 as DNA templates. [Figure 3] This shows the analysis of poly(A) tails by acrylamide gel electrophoresis. 1: Marker, GeneRuler™ 1kb DNA ladder (Thermo Fisher Scientific). Cas9: RT-PCR product against RNA using linearized template DNA with SpCas9. IIS type: RT-PCR product against RNA produced using linearized template DNA with IIS restriction enzyme. PCR: RT-PCR product against RNA produced using template DNA produced by PCR. [Figure 4] This paper shows the expression levels of the reporter protein GFP in Escherichia coli (E. coli) K12, compared to the pTac1 parent variety using an example of GFP expression, comparing it to the basic expression and the expression levels induced by the modified promoters WAC003 and WAC003-LacIDel. [Figure 5] A schematic diagram of the plasmid pCas9_tracrRNA_CRISPR is shown. [Figure 6] This is a schematic diagram of a construct for genome integration using λRed recombination. [Examples]

[0087] Example 1: Plasmid DNA cleavage activity by Cas9 To produce the plasmid, a construct comprising a template sequence, a poly(A) tail, and a target sequence for cleavage by Cas nuclease, as well as a PAM, were first designed. In addition to the GFP mRNA template sequence, the construct encodes a poly(A) tail at the 3' end of the sequence, followed by three additional nucleotides (5'-GTC-3') and a protospacer adjacent motif (PAM) 5'-CGG-3'. The PAM sequence is a Cas9 gRNA recognition sequence for processing. To produce the template DNA sequence, the GFP gene (UniProt: C5MKY7) was amplified using two suitable primers (SEQ ID NOs: 8 and 9) with Phusion polymerase (Thermo Fisher Scientific) in PCR according to a standard protocol. The two primers consisted of, firstly, a T7 RNA polymerase promoter (forward primer, SEQ ID NO: 8) and secondly, the elements: poly(T), 5'GAC3', and PAM (5'-CGG-3') (reverse primer, SEQ ID NO: 9). Next, the PCR-produced organism (SEQ ID NO: 10) was cloned into the pUC18 vector (New England Biolabs) via XbaI and EcoRI cleavage sites. The resulting plasmid pGFP1 (SEQ ID NO: 11) was used as the starting plasmid in the following experiments / examples for the production of linearized template DNA.

[0088] After successfully producing plasmid pGFP1, plasmid DNA was synthesized using E. coli NEB10β (New England Biolabs). This was done by transforming NEB10β with plasmid pGFP1 using the heat shock method according to the manufacturer's instructions. To achieve this, cells and 100 ng of pGFP1 were incubated on ice for 30 minutes, followed by a heat shock at 42°C for 30 seconds. After the regeneration phase (1 hour at 37°C), transformants were selected on selective medium (LB ampicillin (LB-Amp)). The generated clones were grown in 3 mL of culture medium under selective conditions, and the plasmids of the clones were validated by restriction pattern and sequencing. Clones containing the desired plasmid were grown in 50 mL of LB-Amp (shaking, 37°C, overnight) to produce a sufficient amount of plasmid DNA. Depending on the clone, 8-14 OD in a shaking flask was used. 600 (Optical density at 600 nm in water) was obtained. Plasmid DNA was finally isolated and purified from the overnight culture medium (steady phase achieved) using the Gene Jet Plasmid Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0089] In the next step, we designed a template DNA for the in vitro production of guide RNA (SEQ ID NO: 1). To enable the synthesis of guide RNA of a specific length and sequence, a DNA sequence complementary to the above guide RNA was added 3' downstream of the T7 RNA polymerase promoter sequence (SEQ ID NO: 12). This, along with the complementary sequence of the T7 promoter (SEQ ID NO: 13), was synthesized as a long single-stranded primer (Metabion).

[0090] To prepare the guide RNA (SEQ ID NO: 1), these two DNA oligonucleotides (SEQ ID NOs: 12 and 13) were mixed in equimolar amounts (100 pmol), boiled at 95°C for 5 minutes, and then hybridized by slowly cooling to room temperature. The resulting hybrid was then used as a template for synthesizing the guide RNA using an in vitro transcription kit (High Yield T7 RNA Synthesis Kit, Jena Bioscience) according to the manufacturer's instructions. Subsequently, the transcription reaction product was purified using the Monarch RNA Cleanup Kit, New England Biolabs (NEB) according to the manufacturer's instructions, and the concentration was determined by UV absorption.

[0091] After preparing the plasmid pGFP1 (SEQ ID NO: 11) and guide RNA (SEQ ID NO: 1), these two were incubated with Cas nuclease SpCas9 (New England Biolabs) in cleavage buffer (20 mM Hepes pH 7.5; 150 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA). SpCas9 and guide RNA were used in equimolar amounts, and both were used in various excesses relative to the pDNA (1x, 2x, 3x, 4x, 5x, and 10x). For more complex synthesis, SpCas9 and guide RNA were first incubated by themselves (15 minutes at 37°C). Then, pDNA (5 nM) and reaction buffer were added. After incubation at 37°C for 60 minutes, the reaction was stopped by adding 20 μL of 2× formamide buffer (Thermo Fisher Scientific), and the pDNA was analyzed by agarose gel electrophoresis (Figure 1).

[0092] For analysis, the cleaved pDNA was separated on a 1% agarose gel (TAE buffer) and then visualized using Midori Green (Nippon Genetics Europe) on a gel documentation system (BioRad) (see Figure 1). The controls used were uncleaved, unincubated plasmid DNA, uncleaved, incubated (37°C for 1 hour) pDNA, pDNA with cleavage buffer, pDNA with cleavage buffer and 1 mM EDTA, pDNA with 25 nM Cas9 but no guide RNA, pDNA with 25 nM guide RNA but no Cas9, and as a positive control, linearized pDNA using XbaI or NcoI (SEQ ID NO: 11) (used according to the manufacturer's instructions, Thermo Fisher Scientific).

[0093] Figure 1 shows that plasmid DNA pGFP1 is linearized by the Cas9 / guide RNA complex. The marker GeneRuler® 1kb DNA ladder (Thermo Fisher Scientific) was loaded into lane 1. The controls used were: pGFP1 without incubation (lane 2, starting material), pGFP1 aqueous solution with incubation (lane 3, without Cas9 and guide RNA), pGFP1 cleavage buffer solution (lane 4, without Cas9 and guide RNA), pGFP1 cleavage buffer + 1mM EDTA solution (lane 5, without Cas9 and guide RNA), pGFP1 incubated with XbaI (lane 6), pGFP1 incubated with NcoI (lane 7), pGFP1 incubated with 25nM Cas9 (without guide RNA, lane 8), and pGFP1 with 25nM guide RNA (without Cas9, lane 9). Test samples were loaded into lanes 10-15 (1x, 2x, 3x, 4x, 5x, and 10x excess of Cas9 / guide RNA relative to pDNA). In addition, Figure 1 shows that as the amount of Cas9 / guide RNA complex increases, more pDNA is linearized. At a 10x molar excess (lane 15), the degree of linearization obtained was virtually the same as that obtained using conventional type II restriction enzymes. This is illustrated by the reactions using XbaI and NcoI (lanes 6 & 7). Dependence on Cas9 / guide RNA complex formation is shown by lanes 8 and 9, in which case, under a 10x molar excess, either the guide RNA (lane 8) or Cas9 (lane 9) component is omitted. No linearization was observed when using pGFP1 samples without enzymes (lanes 3, 4, and 5).

[0094] Example 2: mRNA synthesis from pDNA cleaved by Cas9 Plasmid DNA pGFP1 (SEQ ID NO: 11) was linearized using SpCas9 and guide RNA (SEQ ID NO: 1) (10x molar excess: see also Figure 1, lane 15) as described in Example 1. The linearized plasmid was then purified using the Gene Jet PCR Purification Kit (Thermo Fisher Scientific). The linearized purified pDNA was then used as template DNA in an in vitro transcription reaction (High Yield T7 RNA Synthesis Kit, Jena Bioscience) according to the manufacturer's instructions to synthesize mRNA. The reaction was carried out at 37°C for 2 hours using 200 ng of template DNA. To remove impurities and kit components, such as enzymes and nucleotides, mRNA was purified from the reaction product using the Monarch RNA Cleanup Kit (NEB) according to the manufacturer's instructions.

[0095] To compare the mRNA production method using the method according to the present invention with conventional methods, namely the linearization of plasmid DNA or the production of PCR products as linear starting DNA using IIS-type restriction enzymes, pGFP2 (SEQ ID NO: 14), produced in the same manner as pGFP1 by amplification of the GFP gene using primers SEQ ID NOs: 9 and 15, was processed and linearized using the IIS-type restriction enzyme BpiI (Thermo Fisher Scientific) according to the manufacturer's instructions. In addition, linear starting DNA was produced by polymerase chain reaction (PCR product) (SEQ ID NO: 16). In this case, a primer with a 60nt poly-A tail (reverse primer, SEQ ID NO: 18) and a primer having a T7 RNA polymerase promoter (forward primer, SEQ ID NO: 17) along with pGFP1 and Phusion DNA polymerase were used as the PCR template (10 ng). The reaction was carried out using the provided buffer system (Thermo Fisher Scientific) according to the manufacturer's instructions. After the reaction, the PCR products were digested with DpnI to remove any remaining methylated plasmid DNA from the PCR reaction. Finally, both the PCR product and the IIS-type linearized plasmid were purified using a PCR purification kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The linear DNA from the PCR reaction and the SpCas9 linearized DNA each had blunt ends, while the IIS-type linearized DNA had a 3' overhang.

[0096] Linear DNA obtained from restriction digestion and PCR was used as template DNA for mRNA synthesis in the same amount as the SpCas9 linearized pDNA (200 ng) used in the in vitro transcription reaction, according to the manufacturer's instructions (High Yield T7 RNA Synthesis Kit, Jena Bioscience). Synthesis was carried out at 37°C for 2 hours. Finally, the mRNA was purified using the Monarch RNA Cleanup Kit (NEB) according to the manufacturer's instructions.

[0097] The prepared mRNA was first analyzed by polyacrylamide gel electrophoresis. In this case, 200 ng of mRNA from each reaction was mixed with RNA loading buffer (Gel Loading Buffer 2, Thermo Fisher Scientific) and boiled at 95°C for 5 minutes. Next, the RNA species were analyzed by separation on a 15% TBE-UREA polyacrylamide gel (Thermo Fisher Scientific; 250V, 50 min). After electrophoresis, the gel was stained with SYBR Green II RNA Gel Stain (Thermo Fisher Scientific) and visualized using a gel documentation system (BioRad) (Figure 2). The control used was 200 ng of in vitro transcription reaction products with uncleaved pGFP1 and pGFP2. pGFP2 was a pGFP1 variant without a PAM sequence (SEQ ID NO: 14). The concentration of the prepared mRNA was determined photometrically using a NanoDrop spectrometer (Thermo Fisher Scientific).

[0098] Figure 2 shows a visualization of the produced mRNA. Lane 1 contains Century Marker® (Thermo Fisher Scientific), and the following lanes contain starting DNA: SpCas9 / guide RNA linearized DNA (Cas9), IIS-type restriction enzyme linearized DNA (IIS-type), DNA from PCR reaction (PCR), and RNA samples corresponding to the uncut plasmids pGFP1 and pGFP2. Figure 2 clearly shows that the major mRNA products obtained from reactions with linearized plasmids (Cas9, IIS-type) and PCR products (PCR) are of the same size. Larger products were evident only in reactions with non-linearized plasmids (pGFP1 and pGFP2). These products originate from "unchecked" in vitro transcription by T7 RNA polymerase. The lack of linearization prevents polymerase "run-off" and "read-through," resulting in a uniform and relatively large product.

[0099] To analyze the poly(A) tail of mRNA, an adapter oligonucleotide (SEQ ID NO: 19) was ligated to the 3' end of mRNA using T4 RNA ligase 1 (NEB). T4 RNA ligase 1 is an enzyme capable of ligating single-stranded RNA. This enzyme ligates a 5'-monophosphate-containing substrate to the 3'-OH end of recipient mRNA. The 3'-OH end is naturally present in the produced RNA. The adapter oligonucleotide was synthesized to contain 5'-monophosphate (Metabion). To prevent self-ligation of the adapter oligonucleotide, the 3' end of the adapter oligonucleotide also contains a synthetic phosphate residue. For ligation, 5 μg of RNA and 40 pmol of the adapter oligonucleotide were used. The reaction mixture (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.5 U RNase inhibitor, 1 mM ATP, 10 U T4 RNA ligase) was incubated at 25°C for 2 hours. Next, the buffer was replaced with water using Amicon (Merck) (100kDa cutoff).

[0100] Subsequently, the obtained adapter ligation product (adapter oligonucleotide + mRNA) was amplified using reverse transcriptase reaction (RT-PCR). For RT-PCR, the Superscript® kit (Thermo Fisher Scientific) was used according to the manufacturer's instructions. For the reaction, specific primers that directly bind to the 5' upstream of poly(A) (forward, SEQ ID NO: 20) and the adapter sequence (reverse, SEQ ID NO: 21) were used. The product obtained from RT-PCR was purified using a PCR purification kit from Thermo Fisher according to the manufacturer's instructions, and the samples were then analyzed by electrophoresis on a 15% polyacrylamide gel. For this purpose, the isolated DNA was stained with Midori Green (Biozym) and visualized using a gel documentation system (BioRad) (see Figure 3).

[0101] Figure 3 shows the analysis of poly(A) tails by acrylamide gel electrophoresis. The marker GeneRuler® 1kb DNA ladder (Thermo Fisher Scientific) was loaded into lane 1. RT-PCR products for RNA produced using DNA templates linearized by different methods (SpCas9 linearization (lane 2), IIS restriction enzyme linearization (lane 3), and PCR products (lane 4)) were separated in the three lanes.

[0102] Figure 3 shows that the same product with exactly the same size distribution of RT-PCR products was obtained for all three RNA samples. Since the source RNAs already have a very high degree of similarity (see Figure 2), it can be inferred that the RT-PCR here had the same or very similar template as the starting material. Therefore, the gel pattern indicates that the method according to the present invention not only enables effective linearization of pDNA, but the pDNA is linearized at the correct predetermined sites, forming the desired uncovered free poly(A) tail.

[0103] Example 3: Promoter design for Cas9-induced expression Constitutive expression of Cas endonucleases, such as Cas9, in vivo in cells can induce nonspecific DNA cleavage events at various sites in both plasmid and bacterial cell chromosomes, and therefore can damage bacterial cells as a plasmid producer. Induced expression of Cas endonucleases, such as Cas9, under a regulated promoter with minimal basic expression is preferred.

[0104] For induced expression of Cas nuclease, and therefore for "activatable" in vivo linearization of plasmid products by adding the inducer during the desired culture time, a limit on the induced expression level is even more preferable. Overly high expression of Cas nuclease within a short time can have adverse effects on the plasmid DNA and the producer cells, as well as those with high basal expression over relatively long periods. Therefore, promoters with low basal expression and moderate post-induced expression levels are particularly preferred.

[0105] The pTac1 promoter (de Boehr, 1983, Proceedings of the National Academy of Sciences 80(1):21-5), a hybrid of the trp and lac promoters, originates from the operator site LacO1, which has only one original Lac operon remaining at position +1 (transcription start site) for the binding of the repressor protein LacI. The repressor protein LacI itself, consisting of a core region with N-terminal and C-terminal subdomains containing an N-terminal "headpiece" DNA binding unit, a hinge region, a lactose-binding region, and a dimerization motif, as well as a C-terminal mini-zipper for tetramerization, is involved in the formation of dimers of dimers. Each LacI dimer binds to the 21 bp double-stranded DNA operator sequence LacO (Lewis 2011. J Mol Biol. 409, 14-27). In natural Lac operons, two double-stranded DNA operator sites (LacO1 and LacO3) are simultaneously bound by an associated LacI tetramer, which forms a so-called "repression loop" in the DNA strand for maximum repression (Oehler et al. 2006, Nucleic Acids Res. 34, 606-612).

[0106] In WAC003 (Accession No. 6) and WAC003-LacIdel (Accession No. 7), the synthetic LacO binding site was inserted via the -35 region of the pTac1 promoter without changing the nucleotide sequences of the -35 and -10 promoter regions, the distance between the two regions (spacer), or the nucleotide sequence of the spacer itself. WAC003 interacts with the native, i.e., tetramerizable LacI repressor protein. In the construct WAC003-LacIdel, the lacI gene is replaced by the variant LacIdel, which generates a different amino acid sequence as a result of a nucleotide frameshift at the C-terminus of the encoded LacI protein, and thus can no longer generate LacI tetramers, but generates LacI dimers.

[0107] The basal expression (repression) and the expression achieved after addition of the inducer (derepression) were examined using the reporter protein GFP. GFP, as a cytoplasmic localization protein, does not stress the cells by itself and is thus extremely suitable as a reference protein for the measurement of expression levels. The gene for GFP was placed under the control of the WAC003 and WAC003-LacIdel promoter constructs, cloned onto a plasmid with a ColE1 replication origin, and this plasmid was transformed into the Escherichia coli (E. coli) K12 strain, and the basal expression and inducible expression in the shaking culture in LB medium were tested as follows.

[0108] From the preculture, the main culture was inoculated to OD 600 = 0.1. After 2 hours, 0.1 mM IPTG was added (induced) or not (basal expression) in correlation with a cell density of approximately OD 600 0.4. After a further 24 hours of culture, the amount of GFP expressed was measured (excitation 380 nm, emission 500 nm) and normalized to the respective basal cell density OD 600 of the culture. The control included was a cell line with the pTac1 promoter (high basal expression, high inducible expression level).

[0109] Figure 4 shows the basal expression and expression levels of the WAC003 and WAC003-LacIdel promoter constructs compared to the Tac1 parent manifold. The WAC003 manifold showed significantly reduced basal expression compared to Tac1. Depression of the WAC003 promoter after the addition of IPTG was similarly significantly reduced (only about 16% GFP / OD compared to Tac1). 600 In the WAC003-LacIdel construct, basal expression is similarly low, and induced expression levels are somewhat higher than in WAC003, but even lower compared to Tac1. Therefore, promoters WAC003 and WAC003-LacIdel are extremely well-suited for the induced expression of Cas nucleases, such as Cas9, and are expressed in vivo for specific linearization of plasmid DNA.

[0110] Example 4: Purification of linear DNA after in vivo induction and incubation of Cas9 To analyze the in vivo functionality of the systems described in Examples 1 and 2, constructs for both the gene encoding SpCas9 (SEQ ID NO: 2) and the guide RNA (SEQ ID NO: 1) were cloned into the same plasmid (pWAC003, SEQ ID NO: 5) under the induced promoter WAC003-LacIdel (SEQ ID NO: 7). The Sp_cas9 gene was amplified from plasmid pCas9-ts1 (SEQ ID NO: 24) for cloning using two SpCas9-specific primers (SEQ ID NOs: 22 and 23). The resulting PCR product was cloned into plasmid pWAC003 (SEQ ID NO: 5) after promoter WAC003-LacIdel (SEQ ID NO: 7) using EcoRI and XbaI. The Cas9 expression cassette of the resulting plasmid pCas9 (SEQ ID NO: 25) was sequenced using specific primers (SEQ ID NOs: 26 and 27) (Sequiserve) to confirm the desired sequence.

[0111] In the in vivo system, guide RNA cannot be produced by run-off transcription as described in Example 2, so adaptation of RNA design is necessary. To produce functional guide RNA, the native form of the RNA to be treated with the CRISPR-Cas9 system must be used. This means preparing a minimal CRISPR array and tracrRNA.

[0112] The tracrRNA sequence (SEQ ID NO: 28) and the CRISPR sequence (SEQ ID NO: 29) were inserted into plasmid pCas9 (SEQ ID NO: 25). In addition to the tracrRNA and CRISPR sequences, the sequences of SEQ ID NO: 28 and 29 each consist of a WAC003-LacIdel promoter sequence and a (5'-3') Rho-dependent stop sequence into which the tracrRNA and CRISPR sequences are embedded. For this purpose, two DNA fragments were first prepared by PCR; the primers used for SEQ ID NO: 28 had SEQ ID NOs: 30-35, and the primers used for SEQ ID NO: 29 had SEQ ID NOs: 36-41. The tracrRNA sequence was cloned into pCas9 using two restriction enzymes, KpnI and XbaI, so that the sequence was 3' downstream of the Cas9 construct. The CRISPR sequence was cloned into plasmid pCas9_tracrRNA (SEQ ID NO: 42), obtained at the 3' position after the tracrRNA sequence via XmaI and XbaI cleavage sites. The correct assembly of the obtained plasmid pCas9_tracrRNA_CRISPR (SEQ ID NO: 43) was verified by sequencing using specific primers (SEQ ID NO: 27).

[0113] Figure 5 shows a schematic diagram of the plasmid pCas9_tracrRNA_CRISPR. In plasmid pCas9_tracrRNA_CRISPR, all three elements—Cas9, tracrRNA, and CRISPR—are under the control of the WAC003-LacIdel promoter and also possess a Rho-dependent termination sequence.

[0114] Plasmid pCas9_tracrRNA_CRISPR (SEQ ID NO: 43) was used to transform *E. coli* NEB10β with pGFP1 (SEQ ID NO: 11). After selection for LB ampicillin / tetracycline, clones were inoculated into 20 mL of LB containing antibiotics (100 mg / L ampicillin or 20 mg / L tetracycline) and incubated overnight at 37°C (200 rpm). The overnight culture was then inoculated into 100 mL of fresh LB medium containing ampicillin and tetracycline (100 mg / L and 20 mg / L, respectively). The resulting major culture was then incubated in 0.8 OD medium. 600nm The cells were incubated at 37°C with shaking as described below. Once the target OD was achieved, IPTG was added to a final concentration of 0.5 mM to simultaneously induce the expression of Cas9, CRISPR cassette, and tracrRNA. Thanks to the simultaneous expression of these elements, plasmid pGFP1 is specifically cleaved. Two hours after induction, the cells were settled by centrifugation. Subsequently, the DNA (linearized pGFP1 and circular pCas9_tracrRNA_crRNA) in the supernatant was isolated and purified using the Gene Jet Plasmid Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0115] The composition of isolated DNA and the proportion of linearized pGFP1 were analyzed by agarose gel electrophoresis and capillary electrophoresis. For agarose gel electrophoresis, isolated DNA was separated on a 1.5% agarose gel and then visualized using Midori Green (Nippon Genetics Europe) and a gel documentation system (BioRad).

[0116] The usefulness of linearized DNA as a template for mRNA production was verified by in vitro transcription as described in Example 2. The obtained RNA was analyzed by polyacrylamide gel electrophoresis, and the poly(A) tail of the mRNA was analyzed using the method described above with T4 RNA ligase 1 and RT-PCR.

[0117] Example 5: Integration of the cas9-CRISPR-tracrRNA construct into the chromosome of a selected Escherichia coli (E. coli) strain. The CRISPR-Cas construct pCas9_tracrRNA_CRISPR (SEQ ID NO: 43), described in Example 4, was incorporated into the chromosome of Escherichia coli (E. coli) NEB10β. The region selected for integration was the intergenetic region of the atpI gene locus. The Cas9-gRNA construct was incorporated using a gene deletion method via λ Red recombination from Datsenko and Wanner (Datsenko et al. 2000, Proc. Natl. Acad. Sci. US A. 97: 6640 - 5).

[0118] To integrate the Cas9-CRISPR-tracrRNA construct into the intergenetic region at the atpI locus in the genome of the Escherichia coli (E. coli) NEB10β strain used, the construct to be integrated was first designed. This construct includes not only the cas9_tracrRNA_CRISPR construct (SEQ ID NO: 43) described in Example 4, but also a chloramphenicol-resistant cassette (cat), two FRT sites adjacent to the cat cassette, and adjacent regions homologous to the integration site. The construct for genome integration by λ Red recombination (SEQ ID NO: 44) is schematically illustrated in Figure 6. The two regions, each 150 bp long and homologous to the integration site, are adjacent to the above construct at their 3' and 5' ends. Between them in the 5'-3' direction are the cas9_tracrRNA_CRISPR construct, the FRT site, the cat cassette, and another FRT site.

[0119] The cat cassette is used to confirm successful integration for later selection. The FRT site is later used to extract the cat cassette again from the genome with the help of recombinase. The homologous region adjacent to the entire construct is 150 base pairs long and is useful for integration at the target site. The construct is integrated into the genome by homologous recombination.

[0120] The completed construct was synthesized using GeneArt and cloned into the pMX vector (GeneArt). From this plasmid, the construct was excised using restriction enzyme (Xhol) and purified using the GeneJet gel extraction kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0121] Homologous recombination for integration was obtained using the λ Red system encoded on plasmid pKD46 (CGSC:7736). NEB10β cells were transformed with the plasmid, and then competent cells were prepared as described by Datsenko and Wanner (Datsenko et al. 2000, Proc. Natl. Acad. Sci. US A. 97: 6640-5). These competent cells were transformed with the purified integration construct. Selection for integration of the chloramphenicol-resistant cassette (cat = chloramphenicol acetyltransferase), as well as Cas9 and guide RNA constructs, into the chromosomes was performed on LB agar plates containing 20 mg / l chloramphenicol. Integration at the desired location in the chromosome was checked / confirmed by PCR using oligonucleotides Check Fwd (SEQ ID NO: 54) and Check Rev (SEQ ID NO: 55) as templates, as well as chromosomal DNA from chloramphenicol-resistant cells. As a negative control, PCR was also performed using chromosomal DNA from the parent strain *E. coli* NEB10β. This method yielded *E. coli* cells in which Cas9 and guide RNA constructs were incorporated into the genome.

[0122] Subsequently, plasmid pKD46 was isolated from cells according to the procedure described by Datsenko and Wanner (see above), and the strain produced by this method was named *E. coli* NEB10β atpI::cas9-CRISPR-tracrRNA-cat. The chloramphenicol-resistant cassette was isolated from the chromosome as specified by Datsenko and Wanner (see above) with the help of plasmid pCP20 (CGSC:7629), which contains the FLP recombinase gene encoding. The chloramphenicol-sensitive strain finally obtained by this method, containing Cas9-CRISPR-tracrRNA, was named *E. coli* NEB10β atpI::cas9-CRISPR-tracrRNA. The successful integration of the construct (SEQ ID NO: 44) was finally verified by sequencing using different primers (SEQ ID NOs: 45-54).

[0123] Next, plasmid pGFP1 (SEQ ID NO: 11) was used to transform a new strain (E. coli NEB10β atpI::cas9-CRISPR-tracrRNA) to test this system. After regeneration and selection (overnight on LB-ampicillin agar plates), colonies were selected and used to first prepare an overnight culture in 20 mL of LB-ampicillin agar (37°C, 200 rpm). This pre-culture was then inoculated into 100 mL of main culture. The resulting main culture was then converted to 0.8 OD. 600nm The following incubation conditions were used. Once the target OD was achieved, IPTG was added to initiate the expression of both Cas9 and guide RNA. After 2 hours under induction conditions, the culture medium was harvested by centrifugation. Subsequently, the plasmid DNA from the cells was isolated and purified using the Gene Jet Plasmid Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0124] The isolated plasmid DNA was analyzed by agarose gel electrophoresis and capillary electrophoresis. For gel electrophoresis, the isolated plasmid DNA was separated according to size and characteristics (linearization, helicalization) using a 1.5% agarose gel, and then visualized using Midori Green (Nippon Genetics Europe) on a gel documentation system (BioRad).

[0125] The usefulness of DNA for mRNA production was checked by in vitro transcription as described in Example 2.

Claims

1. A method for producing a linear double-stranded DNA molecule, wherein the linear double-stranded DNA molecule has a blunt end, and the linear double-stranded DNA molecule has a poly-T sequence at the 5' end of the template strand. (1) On the template chain in the direction from 3' to 5': (i) A template sequence operably linked to an RNA polymerase promoter, (ii) PolyT sequence, (iii)N １ N ２ N ３ (Here, N １ ~N ３ Each of these is independently a nucleotide sequence described by (G, T, A, or C), and (iv) Protospacer adjacent motif (PAM), The steps include preparing a double-stranded circular DNA molecule containing, (2) A step of preparing a guide RNA comprising the 5' end of the polyT sequence (ii), a region complementary to the nucleotide sequence (iii), and a region that interacts with the Cas nuclease, (3) Steps to prepare type II Cas nuclease, (4) The step of contacting the double-stranded circular DNA molecule with the guide RNA and the CAS nuclease, It includes, The method wherein the type II Cas nuclease is Cas9 derived from Streptococcus pyogenes having sequence number 2, or a sequence having at least 90% identity thereto.

2. The method according to claim 1, wherein the polyT sequence (ii) consists of 40 to 250 T nucleotides.

3. The region of the guide RNA that is complementary to the 5'-end of the poly-T sequence (ii) and the nucleotide sequence (iii) has the sequence 5'-G(A) ｎ N ４ N ５ N ６ -3' (where n is a number from 17 to 23, and N ４ to N ６ are each independently G, U, A or C), and the nucleotide N ４ N ５ N ６ is complementary to the nucleotide N １ N ２ N ３ from (iii), the method according to claim 1.

4. The method according to claim 1, wherein the type II Cas nuclease is Cas9.

5. The method according to claim 1, wherein the PAM has the sequence 5'-NGG-3' (wherein N is independently selected from G, C, A and T).

6. The aforementioned polyT sequence (ii) consists of 120 T nucleotides, The region of the guide RNA that is complementary to the 5' end of the polyT sequence (ii) and the nucleotide sequence (iii) is sequence 5'-G(A) ｎ－ N ４ N ５ N ６ -3' (where n is equal to 17, N ４ ~N ６ Each of them independently has G, U, or C, The PAM has the sequence 5'-NGG-3' (where N is independently selected from G, C, A and T), The method according to claim 1.

7. The method according to claim 1, wherein the guide RNA is prepared by expressing a nucleotide sequence encoding the guide RNA.

8. The method according to claim 1, wherein the type II Cas nuclease is prepared by expressing the nucleotide sequence encoding the Cas nuclease.

9. The method according to claim 7 or 8, wherein the nucleotide sequence encoding the guide RNA and / or the nucleotide sequence encoding the Cas nuclease is operably linked to an inducible promoter.

10. The method according to claim 9, wherein the inducing promoter is a lactose-induced promoter.

11. The method according to claim 7 or 8, wherein the nucleotide sequence encoding the guide RNA and / or the nucleotide sequence encoding the Cas nuclease is incorporated into the genome of Escherichia coli (E. coli).

12. The use of type II Cas nuclease for linearization of double-stranded circular DNA molecules, wherein the linear double-stranded DNA molecule is arranged on the template strand in the 3' to 5' direction. (i) A template sequence operably linked to an RNA polymerase promoter, (ii) PolyT sequence, (iii)N １ N ２ N ３ (N １ ~N ３ (Each of these is independently G, T, A, or C.) The nucleotide sequences described by, and (iv) Protospacer adjacent motif (PAM), It includes, The type II Cas nuclease is Cas9 derived from Streptococcus pyogenes having sequence number 2, or a sequence having at least 90% identity thereto.

13. Use for producing a linear double-stranded DNA molecule of a recombinant microorganism whose genome incorporates a nucleotide sequence encoding a guide RNA and / or a nucleotide sequence encoding a type II Cas nuclease, wherein the linear double-stranded DNA molecule has blunt ends and has a poly-T sequence at the 5' end of the template strand, the type II Cas nuclease is Cas9 derived from Streptococcus pyogenes having Sequence ID No. 2 or a sequence having at least 90% identity thereto, and the nucleotide sequence encoding the guide RNA includes a region that interacts with the type II Cas nuclease.

14. The use according to claim 13, wherein the recombinant microorganism is Escherichia coli (E. coli).

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