CRISPR-Cas9-based linearization of plasmid DNA templates

Description

[0001] The invention relates to a method for producing a linear double-stranded DNA molecule, wherein the linear DNA molecule has smooth ends, and wherein the linear DNA molecule has a poly T sequence at the 5' end of the template strand, comprising the steps: (1) Providing a double-stranded circular DNA molecule comprising, on the template strand in the 3' to 5' direction: (i) a template sequence operatively linked to an RNA polymerase promoter, (ii) a polyT sequence, (iii) a nucleotide sequence represented by N1N2N3, wherein N1 to N3 each independently represent G, T, A or C or a nucleotide analogue thereof, and (iv) a protospacer adjacent motif (PAM), (2) providing a guide RNA comprising a region complementary to the 5' end of the polyT sequence (ii) and to the nucleotide sequence (iii), and a region interacting with a Cas nuclease, (3) providing a Cas nuclease type II, (4) bringing the double-stranded circular DNA molecule into contact with the guide RNA and the CAS nuclease.

[0002] Ribonucleic acids (RNAs) play a crucial role in many cellular processes. According to the central dogma of biology, they mediate the translation of DNA into proteins and, with messenger RNAs (mRNAs) and transfer RNAs (tRNAs), play a key role in transcription and translation. But many other processes, especially at the regulatory level—that is, the fine-tuning of biochemical reactions inside and outside cells—are also significantly controlled by RNAs. Therefore, it is not surprising that RNAs are a focus of much research aimed at better understanding these processes.

[0003] In addition to fundamental work on the general understanding of RNAs, recent years have shown how useful RNAs are for therapeutic applications, such as vaccines against SARS-CoV-2. For such applications, it is crucial that suitable methods are available to produce sufficient quantities of mRNA. These methods utilize naturally occurring molecules, such as RNA polymerases, which produce RNAs. The synthetic production of RNAs by DNA-dependent RNA polymerases is a method routinely used in research and industrial applications. This process requires a DNA template, an RNA polymerase (e.g., T7 RNA polymerase), nucleotides such as ATP, CTP, GTP, and UTP, as well as suitable buffer conditions. The polymerase then synthesizes a corresponding complementary RNA, depending on the DNA template. The produced RNAs can be of different types: mRNAs,self-amplifying RNAs (saRNAs), long non-coding RNAs (lncRNAs), guide RNAs (gRNAs), short interfering RNAs (siRNA), micro RNAs (miRNAs), circular RNAs (circRNAs), and others. Especially for shorter RNAs, chemical synthesis can be used industrially. However, for longer RNAs of over 100 nucleotides, such as mRNAs, chemical synthesis is not economical, which is why longer RNAs are usually synthesized enzymatically. in in vitro transcription can be produced.

[0004] In large-scale production, a in vitro- Transcription typically uses plasmid DNA as a DNA template because, unlike PCR-generated DNA templates, plasmid DNA can be produced in the necessary quantities via fermentation. The closed, circular nature of plasmids leads to a theoretically infinitely long RNA transcript during RNA synthesis, but in practice, at least some unintentionally heterogeneous, elongated RNAs are always produced. These elongations negatively impact the efficiency of the transcription process, thereby increasing production costs. Two possible approaches to avoid these unwanted elongated RNA transcripts are described in the literature.

[0005] One solution is the classic use of type II restriction endonucleases. These enzymes recognize a specific DNA sequence and typically make a targeted cut in the double strand of DNA within or near that sequence. Such a cut in the plasmid DNA is used to create a so-called in vitro Run-off transcription allows the RNA polymerase to "drop off" at the double-strand break and re-bind to a promoter, initiating a new synthesis cycle. Classical type II restriction enzymes are typically used for such a cut in the DNA template. Type IIP restriction enzymes cut DNA at defined positions within their recognition sequences. Examples of type IIP restriction enzymes include EcoRI and [missing example]. XbaI. Alternatively, enzymes of the subgroup type IIS, which cut outside their recognition sequences, can also be used. Both classes of type II restriction enzymes (type IIP and type IIS) can be used to generate linear DNA fragments from circular plasmid DNA, which can then be used for more efficient RNA synthesis. It is important to note that the DNA template should not contain any further recognition sequences for the respective restriction enzymes used. This results in a limitation of the template sequence or a more complex design. This can affect individual base pairs, but also entire codons of the DNA template or domains of the target proteins, such as motifs or structures. Additionally, many type II restriction enzymes, especially type IIS enzymes, process the DNA with an overhang, resulting in so-called 5' or 3' sticky ends.A 3' overhang is particularly problematic, as the promiscuity of RNA polymerase can lead to longer transcripts than originally intended. To prevent these unwanted transcripts, the overhang would have to be removed after the restriction step. This significantly increases the number of steps involved in planning and generating the DNA template, thus increasing the overall development effort.

[0006] The second solution for avoiding excessively long RNA transcripts is the use of Rho-independent termination signals. Unlike the use of restriction endonucleases, the DNA template is not linearized here. Rho-independent terminators are typically secondary structures within the synthesized target RNA and prevent the formation of excessively long RNA transcripts by halting synthesis. After successful termination, the polymerase, similar to run-off transcription, can bind to a promoter again and initiate a new synthesis cycle. This maintains a high transcription rate, but also results in the addition of undesired sequence regions. z.B. the terminator sequence. In addition to RNA-cutting enzymes, other methods can be used to remove these unwanted elements. z.B. RNases, also known as catalytically active nucleic acids, are used. These include, for example, DNAzymes, but also so-called ribozymes. Ribozymes are structured RNA molecules which in trans (coming from outside) or in cis (Starting from the sequence) RNAs are cut at defined sites. This usually requires a specific RNA structure and a divalent metal ion, typically Mg²⁺. This results in a nucleophilic attack on the target sequence, causing it to be cleaved and, in the case of RNA synthesis, allowing excess elements to be removed. Since T7 RNA polymerase also relies on Mg²⁺ ions, ribozyme activity within the synthesis reaction will influence the efficiency of the in Vitro transcription is subject to competition for Mg²⁺ ions. This necessitates further optimization of the reaction conditions, which can lead to efficiency losses. When designing corresponding sequences with a cis-ribozyme, care must be taken to ensure that a ribozyme followed by at least one terminator sequence is incorporated into the target RNA. Consequently, the entire process for producing DNA templates for mRNA synthesis becomes more expensive and therefore less economical.

[0007] For the enzymatic production of functional mature mRNAs and saRNAs intended to be active in mammalian cells, it is essential that the RNA possesses a poly(A) attachment. This poly(A) attachment increases the stability of the RNA and also plays a crucial role during translation in the eukaryotic target cell. The poly(A) attachment is recognized and bound in the target cell by poly(A)-binding proteins. These bound proteins facilitate the interaction of the 3' end of the mRNA with the proteins of the translation machinery at the 5' end of the mRNA. This complex interplay increases not only the stability of the RNA but also its translation efficiency. This results in a key role for the poly(A) attachment within mature mRNA. To represent the most natural mRNA or saRNA possible, the poly(A) attachment should be as long as possible (> 100 A nucleotides) and ideally homogeneous (without other bases such as C, G, or U).This is then referred to as an unmasked poly A appendage at the 3' end of the RNA.

[0008] Two methods for producing RNA molecules with a uniform poly(A) attachment are currently common. One method involves generating the poly(A) attachment using a poly(A) polymerase after the synthesis of the target RNA sequence. In this enzymatic step, the synthesized RNA is first purified before a poly(A) attachment is synthesized by adding a poly(A) polymerase and ATP as a substrate. The duration of the reaction determines the average length of the poly(A) attachments. This enzymatic synthesis generally works very well but also has some drawbacks. For example, it is not possible to produce a precisely defined poly(A) attachment length with this technique, resulting in a product with inhomogeneous RNA molecules of normally distributed lengths. Therefore, for industrial use of the RNA molecules produced in this way, further processing steps for enrichment or purification of the RNAs are necessary, which reduces yields and increases production costs.

[0009] In contrast, a method involves directly encoding the poly A subset using the DNA template, which serves as the blueprint for RNA synthesis. For polyadenylation in this method, the template DNA is designed to ensure that the double-stranded DNA on the template strand contains the necessary poly T motif. The advantage of this approach is that it produces a significantly more defined product with a poly A subset of uniform length in a single reaction step.

[0010] When PCR products are used as templates for the synthesis of RNA molecules with homogeneous and unmasked poly(A) attachments, the poly(A) attachment can be directly encoded in the primers used for PCR. A reverse primer can be used to prepare a DNA template via PCR; this primer contains, in addition to the complementary sequence at the 3' end, a sequence of up to 120 or more adenines. In PCR, this attachment is added to the coding DNA and thus represents the in vitro- Transcription is available. Since PCR reactions can usually only be performed technically in small volumes up to a few hundred microliters, several hundred PCR reactions would be required for large-scale processing to RNA. Scaling up the PCR approach through a so-called scale-out, in which several reactions are carried out in parallel, is currently not economically viable on an industrial scale due to the high labor and cost requirements.

[0011] When plasmid DNA is used as a template for the in If in vitro transcription of RNA is used, fermentative production of the plasmids, purification of the pDNA, and subsequent linearization of the pDNA are necessary. After linearization, the linearized pDNA must be purified again to obtain the purest possible DNA template for the in to provide in vitro transcription, thereby increasing the complexity and cost of the procedure. As previously noted, a disadvantage of using type IIP restriction enzymes is that these enzymes do not produce a free, unmasked poly A appendage, since the recognition sequence of type IIP restriction enzymes almost always includes other nucleotides such as C, G, or T. While the use of type IIS enzymes could remedy this, as they cleave outside their recognition sequence and thus create the desired unmasked poly A appendage, the use of type IIS enzymes typically leads to 3' or 5' overhangs, which would first have to be degraded as described above.

[0012] Besides using restriction enzymes, it is also possible to use ribozymes to linearize the template DNA. These can also be designed to produce a homogeneous and free poly(A) attachment. Ribozymes are preferably designed to cleave directly at the end of the poly(A) sequence. The disadvantage of ribozymes is that... trans Ribozymes produce another RNA, or in the case of cis Ribozymes require the production of a significantly longer RNA molecule. This approach increases the production effort and, consequently, the costs.

[0013] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) CRISPR-associated (Cas) nucleases have been identified as components of bacterial immune systems. Because Cas nucleases can precisely cut DNA elements at a specific location, CRISPR-Cas systems have been further developed as efficient gene-editing tools, primarily for genome editing applications, leading to a revolution in molecular 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).

[0014] When a bacterial cell is infected by a foreign nucleic acid, such as phage DNA, transposons, pDNA, or RNA, the bacterium mounts an immune response to eliminate the foreign DNA or RNA. What is special about CRISPR-Cas immunity is that it is adaptive immunity, meaning the bacterial cell learns this type of immune response. During an infection, a short DNA fragment of the foreign nucleic acid is used by the immune system, enabling a faster response to a similar infection later on. This DNA fragment is inserted within the genetic locus for the CRISPR-Cas system in the CRISPR array and is used there as a spacer This spacer sequence later becomes part of the guide RNA (gRNA) or CRISPR RNA (crRNA) and thus contributes to sequence specificity. During this bacterial immune response, the Cas nuclease is directed by the crRNA to a specific target sequence on the foreign nucleic acid. For sequence recognition, the crRNA must be complementary to the foreign target DNA, and a short signal sequence in close proximity to the target DNA is required. Protospacer Adjacent Motif (PAM). The PAM sequence consists of a different number of specific nucleotides depending on the CRISPR-Cas system and is the initial recognition sequence for the Cas nuclease on the DNA to be cut. For example, the Cas nuclease Cas9 recognizes Streptococcus pyogenes (SpCas9) the PAM sequence 5'-NGG-3'. Without the PAM sequence in close proximity to the target sequence, no cleavage of the foreign DNA occurs. After recognition by the ribonucleoprotein complex of Cas nuclease and crRNA, the foreign target sequence is specifically cleaved, and subsequently, the foreign DNA can be degraded by other cellular nucleases. Only the appropriate combination of PAM sequence and target sequence in close proximity leads to the cleavage activity of the Cas nuclease. Since a specific combination of PAM and target sequence is generally not present in the bacterial genome, an autoimmune reaction, i.e., the cleavage of the bacteria's own DNA, is prevented.

[0015] There are different CRISPR-Cas systems, which differ primarily in the type and number of proteins required for the CRISPR complex to bind and cut at the correct location in the target sequence. Type II-A systems, which use the Cas9 protein as an effector nuclease for interference with the target DNA, are particularly well understood. Besides the eponymous CRISPR array, which encodes the various crRNAs, type II systems include a trans-activating RNA (tracrRNA), the adaptation proteins Cas1, Cas2, and Cas4, and the effector nuclease Cas9. The tracrRNA forms a hybrid with the crRNA encoded by the CRISPR array. This hybrid is then specifically bound by Cas9. The crRNA portion of the hybrid construct guides Cas9 to the target DNA. The two-part system of crRNA and tacrRNA is relatively cumbersome for use in molecular biology; therefore, the crRNA and the tacrRNA are often connected with a linker.This resulting RNA is called sgRNA (single guide RNA).

[0016] One object of the invention is to provide a simplified or improved method for linearizing circular DNA molecules that can be used as a DNA template for enzymatic RNA synthesis. For this purpose, the linearized DNA molecule should have smooth ends and a polyT sequence at the 5' end of the template strand. In particular, the method should provide linearized DNA molecules that enable the synthesis of RNAs with a homogeneous and unmasked polyA appendage. Another object of the invention is to provide a method that requires fewer steps than the methods disclosed in the prior art. Another object is to provide a method that yields linear DNA molecules with improved homogeneity with respect to the length of the polyA appendage. A further object is to provide a method that is more efficient or...can be carried out more cost-effectively than the methods described in the prior art.

[0017] The problem is solved by a method for producing a linear double-stranded DNA molecule, wherein the linear DNA molecule has smooth ends, and wherein the linear DNA molecule has a poly T sequence at the 5' end of the template strand, comprising the steps: (1) Providing a double-stranded circular DNA molecule comprising, on the template strand in the 3' to 5' direction: (i) a template sequence operatively linked to an RNA polymerase promoter, (ii) a polyT sequence, (iii) a nucleotide sequence represented by N1N2N3, wherein N1 to N3 each independently represent G, T, A or C or a nucleotide analogue thereof, and (iv) a protospacer adjacent motif (PAM), (2) providing a guide RNA comprising a region complementary to the 5' end of the polyT sequence (ii) and to the nucleotide sequence (iii), and a region interacting with a Cas nuclease, (3) providing a Cas nuclease type II, (4) bringing the double-stranded circular DNA molecule into contact with the guide RNA and the CAS nuclease.

[0018] An application of CRISPR-Cas systems in the field of linearization of plasmid DNA, especially as a template for a in The vitreous transcription of RNA is not yet known. By combining a guide RNA, a Cas nuclease (e.g., Cas9), and a double-stranded circular DNA molecule, a CRISPR-Cas system can be used for the efficient and homogeneous linearization of plasmid DNA.

[0019] Due to the flexible design of the guide RNA, the linearization method can be used flexibly for all double-stranded circular DNA molecules, allowing for a specific cut at a pre-selected location of the DNA.

[0020] Due to the flexible design options of the PAM, the linearization process can be flexibly carried out with different Cas nucleases.

[0021] Type II Cas nucleases, such as Cas9, form so-called "smooth" or "blunt" DNA ends when cutting the double strand in the target molecule. d.h. DNA ends without 3'- or5'- Overhang. Such DNA templates with smooth ends are particularly well-suited for RNA synthesis. In mRNA synthesis, it is desirable that the resulting mRNA possesses a homogeneous and unmasked poly(A) attachment of as defined a length as possible. The more homogeneous the poly(A) attachments of the mature mRNA are, d.h. The fewer bases other than adenine are present, the simpler and more efficient the binding of the poly-A-binding protein. The binding of this protein, in turn, is crucial for effective translation of the mature mRNA within the target cell. Negative effects of mRNAs that do not terminate directly with adenine residues or with different bases, such as those that arise when using many type II restriction enzymes, can thus be avoided. The inventive method therefore enables the production of linear DNA templates that are particularly well-suited for the production of natural, mature RNA, for example, mRNA, which offers advantages in therapeutic applications. z.B. improved stability, longer half-life, or higher translational efficiency.

[0022] The method according to the invention offers a high degree of freedom in sequence design and absolute sequence independence. d.h. There are no restrictions on nucleotides, sequence and structure motifs.

[0023] The inventive, CRISPR-Cas-based method is independent of methylations and other modifications of the DNA, as is the case with many restriction enzymes.

[0024] According to the invention, a linearization of the double-stranded circular DNA molecule, d.h. The linearization of the DNA template can occur during the replication of the plasmid DNA, for example, within the bacterial production cell or before cell disruption. This offers the advantage of reducing the number of process steps. Thus, at the end of pDNA fermentation, linearization by a Cas nuclease, such as Cas9, can take place directly in the cell. After completion of fermentation and intracellular linearization, the linear DNA can be purified using conventional methods, eliminating the need for separate processing of the double-stranded circular DNA molecule. The linear DNA template can then be used directly in a in vitro- Transcription can be used to produce RNA, for example mRNA with an unmasked poly A appendage.

[0025] The linear double-stranded DNA molecule produced according to the invention has smooth ends and a poly T sequence at the 5' end of the template strand. Such linear DNA molecules are particularly well suited as DNA templates for a in In vitro transcription. During the in In vitro transcription is an enzymatic catalysis in which in vitro RNA is produced by synthesizing the complementary RNA from a DNA template using a DNA-dependent RNA polymerase. The synthesis also requires ribonucleotides (NTPs), a buffer system (e.g., TRIS-HCl), magnesium ions (e.g., MgCl₂), and optionally an RNase inhibitor, an inorganic pyrophosphatase, and spermidine. Typically, monomeric RNA polymerases are used, which are usually bacteriophage enzymes. The most commonly used polymerases are, for example, T7 RNA polymerase (Uniprot: P00573) and T3 RNA polymerase (Uniprot: Q778M8). Definitions

[0026] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as they would normally be understood by a person skilled in the field of the present invention. Each technical feature mentioned in the following definitions can be applied to any embodiment of the invention.

[0027] The terms nucleic acid sequence, nucleotide sequence, DNA sequence, and RNA sequence are familiar to those skilled in the art and refer to a specific and individual sequence of nucleotides. Within the scope of this invention, the terms DNA and RNA are used synonymously.

[0028] 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 naturally, chemically, or biochemically modified, non-natural, or derivatized nucleotide bases.

[0029] A nucleotide analogue refers to a chemical compound that is structurally and functionally similar to the nucleotide; that is, the nucleotide analogue can be recognized as a substrate by a polymerase. In this context, nucleotide analogues specifically include native and non-native variants of the naturally occurring nucleotides adenosine, cytosine, thymidine, guanosine, and uridine. Examples include chemically derivatized nucleotides with non-native functional groups that are added to or removed from the naturally occurring nucleotide, or that replace the naturally occurring functional groups of a nucleotide. Examples of nucleotide analogues include N1-methylpseudouridine, 5-methoxyuridine, 5-methylcytidine, pseudouridine, N4-acetylcytidine, and N6-methyladenine.

[0030] A poly T sequence as used herein is understood to be a sequence of thymine nucleotides, preferably containing no other nucleotides, i.e., no adenine, guanine, or cytosine.

[0031] A poly A sequence as used herein is understood to be a sequence of adenine nucleotides, preferably containing no other nucleotides, i.e., no thymine, guanine, or cytosine.

[0032] A poly-T sequence or a poly-A sequence can be segmented into two or more distinct segments, each containing at least approximately 40 thymine or adenine nucleotides, respectively. Each segment may be interrupted by sequences of approximately 10–20 nucleotides in length that are not poly-T or poly-A sequences and may also contain glycosaminoglycans (G), cyanocobalamin (C), and uranium (U). Such segmentation can improve the stability of the poly-T or poly-A sequence.

[0033] An unmasked poly A appendage of an RNA is understood here to be a terminal poly A sequence in which the sequence of adenine nucleotides at the 3' end of the RNA is followed by no other nucleotides besides adenine, i.e., no cytosine, uracil, or guanine.

[0034] A poly A attachment of an RNA from a in In vitro transcription is considered homogeneous if, in addition to the uncovered 3' end, the poly(A) exhibits a narrow size distribution around a defined target length. Preferably, the average deviation from the target length is no more than 10% based on the number of nucleotides, more preferably no more than 5%, and most preferably less than 5%. A segmented poly(A) appendage is considered homogeneous if the sum of all segments exhibits the aforementioned average deviation from the target length of all segments. Segmentation can stabilize the entire poly(A) appendage. Even in the case of a segmented poly(A) appendage of an RNA, it is advantageous for the 3' segment of the poly(A) appendage to be uncovered to ensure high translation efficiency.

[0035] A template strand of double-stranded DNA is the DNA strand that is read by an RNA polymerase during transcription in order to synthesize the RNA.

[0036] A template sequence, or template DNA, or DNA template, is the DNA sequence that serves as the basis for a in vitro- Transcription is used. This sequence serves as a template for a DNA-dependent RNA polymerase to synthesize an RNA molecule according to this sequence.

[0037] A template sequence can include a nucleic acid that codes for an antigen, for example a tumor antigen, a viral or bacterial antigen, a therapeutic protein, or for other proteins such as growth factors or transcription factors.

[0038] As used herein, a promoter sequence or promoter is a DNA regulatory region capable of binding an RNA polymerase and triggering the transcription of a downstream (in the 3' direction) coding or non-coding sequence. Suitable promoters can be derived from any organism, including prokaryotic and eukaryotic organisms.

[0039] As used herein, "operatively associated with a promoter" means that the promoter causes or regulates the transcription of DNA encoding a gene, such as the gene encoded by the template sequence, the gene encoding the guide RNA, or the 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 is introduced. In one embodiment, the promoter is an inducible promoter, i.e., the promoter is regulated to enable inducible transcription of a gene, such as the gene encoding the guide RNA or the gene encoding a type II Cas nuclease.

[0040] The promoter can be a constitutively active promoter, meaning a promoter that is constitutively in an active state, or it can be an inducible promoter, meaning a promoter whose state is controlled by an external stimulus, i.e., switched from an inactive to an active state. A stimulus or inducer induces, or activates, the expression of a target gene. This method is preferred when the expression or overexpression of a target gene has negative effects on the production organism or on a process. Such an external stimulus could be, for example, a specific temperature, a compound, or a protein.

[0041] Examples of inducible promoters include the Lac promoter, which is inducible by IPTG (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-inducible promoter (Kelly et al., ACS Synth. Biol.; 2016, 5 Pages 1136-1145), and the arabinose promoter, which is inducible by arabinose (Guzman et al., J Bacteriol. 1995, Jul;177(14):4121-30.).

[0042] The term Protospacer Adjacent Motif PAM describes a signal sequence of approximately 2 to 8 base pairs that represents the specific recognition site for a Cas nuclease or for a multiprotein Cas effector complex (e.g., CASCADE or CMR) on the DNA to be cut. Without a PAM signal sequence, the target DNA is not cut. The length and base sequence of the PAM sequence vary depending on the CRISPR-Cas system and Cas nuclease, as well as the organism in which the Cas nuclease naturally occurs. For example, the PAM sequence associated with the Cas9 nuclease of Streptococcus pyogenes is associated with (SpCas9), 5'-NGG-3', where "N" is any nucleotide, i.e., can be A, T, G, or C, followed by two guanine nucleotides. Further species and their associated PAM sequences are known to those skilled in the art and described in the prior art (Fonfara et al. Nucleic Acids Research, 2014, Vol. 42, No. 4, pp. 2577-2590).

[0043] A guide RNA, gRNA or guide Guide RNA is a specific RNA sequence to which a Cas nuclease can bind, directing it to the template DNA. The guide RNA hybridizes specifically to a particular target site on the template DNA, where the Cas nuclease cleaves the template DNA. The guide RNA can be bipartite, a hybrid of crRNA and tracrRNA. Alternatively, the guide RNA can be a single sgRNA. (single guide RNA), in which the essential regions of crRNA and tracrRNA are linked together, resulting in a continuous chimeric RNA. An sgRNA mimics the hairpin structure of the crRNA::tracrRNA duplex and is also capable of binding a Cas nuclease and directing it to the target sequence on the template DNA. The guide RNA is designed based on the sequence information of the template DNA to be cut.

[0044] Complementary or complementarity, as used herein, describes the relationship between two DNA or RNA nucleic acid regions which, due to their nucleobases, match each other according to Watson-Crick and form hydrogen bonds with each other, i.e., hybridize. Standard Watson-Crick base pairings are known in the prior art, for example: adenine (A) pairs with thymidine (T), adenine (A) pairs with uracil (U), and guanine (G) pairs with cytosine (C).

[0045] Homologous genes or sequences are defined here as genes or sequences whose DNA sequences are at least 70%, preferably at least 80%, preferably at least 90%, and most preferably at least 95% identical. The degree of DNA identity is determined by the "nucleotide blast" program, available at http: / / blast.ncbi.nlm.nih.gov / , which is based on the blastn algorithm. The algorithm parameters used for aligning two or more nucleotide sequences were preset. The preset 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 preset scoring parameters are: Match / Mismatch Scores = 1, -2; Gap Costs = Linear.

[0046] A homologous amino acid sequence is defined as a sequence that is at least 70%, preferably at least 80%, preferably at least 90%, and most preferably at least 95% identical, with each modification in the homologous sequence being selected from insertion, addition, deletion, and substitution of one or more amino acids. The program "protein blast," available at http: / / blast.ncbi.nlm.nih.gov / , is used to compare protein sequences. This program utilizes the blastp algorithm. The predefined parameters were used as algorithm parameters for aligning two or more protein sequences. The predefined 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 matrix adjustment. Detailed description of the invention

[0047] In one aspect, the present invention relates to a method for producing a linear double-stranded DNA molecule, wherein the linear DNA molecule has smooth ends, and wherein the linear DNA molecule has a poly T sequence at the 5' end of the template strand, comprising the steps: (1) Providing a double-stranded circular DNA molecule comprising, on the template strand in the 3' to 5' direction: (i) a template sequence operatively linked to an RNA polymerase promoter, (ii) a polyT sequence, (iii) a nucleotide sequence represented by N1N2N3, wherein N1 to N3 each independently represent G, T, A or C or a nucleotide analogue thereof, and (iv) a protospacer adjacent motif (PAM), (2) providing a guide RNA comprising a region complementary to the 5' end of the polyT sequence (ii) and to the nucleotide sequence (iii), and a region interacting with a Cas nuclease, (3) providing a Cas nuclease type II, (4) bringing the double-stranded circular DNA molecule into contact with the guide RNA and the CAS nuclease.

[0048] For the process according to the invention, a specifically designed, double-stranded, circular DNA molecule is provided. The circular DNA molecule comprises, on the template strand in the 3' to 5' direction, a template sequence operatively linked to an RNA polymerase promoter, a polyT sequence followed by a nucleotide sequence represented by N1, N2, N3, wherein N1 to N3 each independently represent G, T, A, or C, or a nucleotide analog thereof, and a Protospacer Adjacent Motif (PAM).

[0049] The RNA polymerase promoter is preferably a recognition sequence for a DNA-dependent RNA polymerase, for example the T7 RNA polymerase promoter, the SP6 RNA polymerase promoter or the T3 RNA polymerase promoter.

[0050] The template sequence comprises, for example, a nucleic acid that codes for an antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen. In another example, the template sequence comprises a nucleic acid that codes for a therapeutic protein or for another protein such as a growth factor or transcription factor.

[0051] The poly-T sequence on the template strand of the double-stranded circular DNA molecule consists, for example, of between approximately 40 and approximately 250 T nucleotides. Preferably, the poly-T sequence consists of between approximately 100 and approximately 140 T nucleotides, and more preferably of approximately 120 T nucleotides.

[0052] In the nucleotide sequence represented by N1 N2 N3, N1 to N3 each independently represent G, T, A, or C, or a nucleotide analogue thereof. Preferably, N1 to N3 each independently represent G, T, A, or C. In a particularly preferred embodiment, N1 to N3 each independently represent G, A, or C.

[0053] There is also a [missing information] on the template strand of the double-stranded circular DNA molecule. Protospacer Adjacent Motif (PAM). Preferably, the PAM immediately follows the nucleotide sequence N1 N2 N3.

[0054] The PAM can be any selected PAM, depending on the specific Cas nuclease to be used in the process according to the 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, wherein N is independently selected from G, C, A, and T, respectively, and wherein the PAM sequences are shown in the 5'-3' direction. In a preferred embodiment, the PAM comprises the sequence 5'-NGG-3', wherein N is independently selected from G, C, A, and T. In another preferred embodiment, the PAM has the sequence 5'-NGG-3', wherein N is independently selected from G, C, A, or T. In a further preferred embodiment, the PAM is selected by a Cas9 nuclease. Streptococcus identified.

[0055] The target site where the circular double-stranded DNA is to be cut is preferably located 3 nucleotides 5' from the PAM.

[0056] The guide RNA for the method according to the invention is designed such that it has a sequence at its 5' end which is complementary to the template strand of the double-stranded circular DNA molecule. This complementarity region consists, for example, of about 20 nucleotides that are complementary to about 20 consecutive nucleotides of the template strand.

[0057] The guide RNA includes a region complementary to the 5' end of the poly T sequence (ii) and to the nucleotide sequence (iii). Preferably, the region of the guide RNA complementary to the 5' end of the poly T sequence (ii) and to the nucleotide sequence (iii) has the sequence 5'-G(A) n -N 4 N 5 N 6 -3'. Here, n is a number between 17 and 23, preferably a number between 17 and 20, and preferably n is equal to 17.

[0058] N 4 to N 6 each independently represent G, U, A, C or a nucleotide analogue thereof, preferably N 4 to N 6 each independently represent G, U, C or a nucleotide analogue, particularly preferably N 4 to N 6 each independently represent G, U or C.

[0059] The complementarity sequence within the guide RNA, for example, has the sequence 5'-GAAAAAAAAAAAAAAAANNN-3', where N is any nucleotide independently. Preferably, the method is characterized in that the guide RNA is SEQ ID NO:1.

[0060] The complementarity of the region of the guide RNA to the 5' end of the polyT sequence (ii) and to 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%, particularly preferably 100%.

[0061] The nucleotides N4, N5, and N6 of the guide RNA are complementary to the nucleotides N1, N2, and N3 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 of nucleotides N4, N5, and N6 and nucleotides N1, N2, and N3 is 100%. Preferred complementary base pairings include, for example, G and C, A and T, and A and U. Complementary sequences include, for example, CAG for N1, N2, and N3 and GUC for N4, N5, and N6.

[0062] Furthermore, the guide RNA includes a region that interacts with a Cas nuclease. Preferably, the guide RNA includes a region that interacts with a type II Cas nuclease.

[0063] Furthermore, a Cas nuclease of type II is provided in the process according to the invention. Any Cas nuclease of type II can be used. For example, a Cas nuclease of type II is used. Streptococcus pyogenes (SpCas9; Uniprot Q99ZW2), Streptococcus mutans (SmCas9; Uniprot Q8DTE3), Streptococcus thermophilus (StCas9; Unitprot G3ECR1), Staphylococcus aureus (SaCas9; Uniprot J7RUA5), Francisella novicida (FnCas9; Uniprot A0Q5Y3), Campylobacter jejuni (CjCas9; Uniprot A0A698TVI8), Neisseria meningitids (MnCas9; Uniprot C9X1G5), Pasteurella multocida (PmCas9; AddGene#68703; https: / / www.addgene.org / 68703 / ). A type II Cas nuclease is preferred. Streptococcus spec. provided. A type II Cas nuclease is preferred. Streptococcus pyogenes provided. The most preferred Cas II nuclease, Cas9, is derived from Streptococcus pyogenes used.

[0064] Cas9 is preferably used as the type II Cas nuclease. For example, Cas9 is selected from Streptococcus pyogenes with SEQ ID NO:2 or a sequence that is at least 70% homologous to it, preferably at least 80% homologous, more preferably at least 90% homologous, more preferably at least 95% homologous, and particularly preferably at least 98% homologous; Staphylococcus aureus with SEQ ID NO:3 or a sequence that is at least 70% homologous to it, preferably at least 80% homologous, more preferably at least 90% homologous, more preferably at least 95% homologous, and particularly preferably at least 98% homologous, or Streptococcus thermophilus with SEQ ID NO:4 or a sequence that is at least 70% homologous to it, preferably at least 80% homologous, more preferably at least 90% homologous, more preferably at least 95% homologous, and most preferably at least 98% homologous.

[0065] In a preferred embodiment, the Cas II nuclease is Cas9 from Streptococcus pyogenes with SEQ ID NO:2 or a sequence that is at least 70% homologous to it, preferably at least 80% homologous, more preferably at least 90% homologous, more preferably at least 95% homologous, and most preferably at least 98% homologous.

[0066] If, in the inventive method, Cas9 is used as the Cas nuclease, S. pyogenes If Cas9 is used, then 5'-NGG-3' is preferably used as the PAM. S. mutans If a Cas9 variant is used, then 5'-NGG-3' is preferably used as the PAM. S. thermophilus If Cas9 is used, then 5'-NGGNG-3' or 5'-NNAAAAW-3' is preferably used as the PAM. Francisella novicida If Cas9 is used, then 5'-NG-3' is preferably used as the PAM. Campylobacter jejuni If Cas9 is used, then 5'-NNNNNACA-3' is preferably used as the PAM. Neisseria menigitidis If Cas9 is used, then 5'-NNNNGATT-3' is preferably used as the PAM. Pasteurella multocida If the PAM used is 5'-GNNNCNNA-3', then the present invention is preferably used, and the present invention is not limited to these examples.

[0067] 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 smooth ends, and wherein the linear DNA molecule has a poly T sequence at the 5' end of the template strand, comprising the steps: (1) Providing a double-stranded circular DNA molecule comprising on the template strand in the 3' to 5' direction: (i) a template sequence operatively linked to an RNA polymerase promoter, (ii) a polyT sequence, wherein the polyT sequence consists of about 120 T nucleotides, (iii) a nucleotide sequence represented by N1N2N3, wherein N1 to N3 each independently represent G, T, A or C or a nucleotide analogue thereof, and (iv) a PAM having the sequence 5'-NGG-3', wherein N each is independently selected from G, C, A and T, (2) Providing a guide RNA comprising a region complementary to the 5' end of the polyT sequence (ii) and to the nucleotide sequence (iii), and a region interacting with a Cas nuclease, wherein the Region which is complementary to the 5' end of the poly T sequence (ii) and to the nucleotide sequence (iii) has the sequence 5'-G(A) n N 4 N 5 N 6 -3', where n is equal to 17,wherein N 4 to N 6 each independently represents G, U or C, (3) providing a Cas type II nuclease, wherein the Cas type II nuclease Cas9 is composed of , Streptococcus pyogenes with the SEQ ID NO:2 or a sequence that is at least 70% homologous to it, (4) Bringing the double-stranded circular DNA molecule into contact with the guide RNA and the CAS nuclease.

[0068] In the process according to the invention, the guide RNA can be provided as an RNA molecule. According to the invention, the guide RNA can also be provided by expressing a nucleotide sequence that encodes the guide RNA. Preferably, the guide RNA is provided by expressing a nucleotide sequence that encodes the guide RNA.

[0069] In the inventive process, the Cas nuclease type II can be provided as a protein. According to the invention, the provision of the Cas nuclease type II can also be achieved by the expression of a nucleotide sequence encoding the Cas nuclease type II. Preferably, the Cas nuclease type II is provided by the expression of a nucleotide sequence encoding the Cas nuclease type II. Particularly preferably, both the guide RNA and the Cas nuclease are provided by the expression of a nucleotide sequence. The nucleic acid encoding the guide RNA and / or the Cas nuclease can be localized on an expression plasmid. For example, the nucleic acid encoding the guide RNA can be localized on one expression plasmid, and the nucleic acid encoding the Cas nuclease can be localized on a separate expression plasmid.In another example, the nucleic acid encoding the guide RNA and the nucleic acid encoding the Cas nuclease are located on the same expression plasmid.

[0070] For example, an expression plasmid containing Cas9 and a guide RNA can be generated as follows. During cloning, a Cas9 nuclease can first be cloned into a suitable expression plasmid, and then, for example, a tracrRNA construct and a minimal CRISPR array can be cloned in the 5' to 3' direction. This allows, for example, the natural formation of a tracrRNA::crRNA duplex of a guide RNA. A suitable expression vector is, for example, pWAC003 (SEQ ID NO:5). low-copy Plasmid with approximately 10 copies per cell based on the pACYC replication origin.

[0071] According to the invention, the double-stranded circular DNA molecule is brought into contact with the guide RNA and the Cas nuclease. This contact can, for example, in vitro In an embodiment wherein the provision of the guide RNA and / or the Cas nuclease type II is effected by the expression of a nucleotide sequence encoding the guide RNA and / or the Cas nuclease type II, the contacting preferably takes place in a cell, in particular in a bacterial cell, which is suitable for amplifying plasmid DNA.

[0072] Constitutive expression of a Cas endonuclease, such as Cas9, can cause nonspecific DNA cleavage events at various locations within the plasmid product, i.e., within the double-stranded circular DNA molecule, or within the chromosome of the bacterial cell used. Early processing of the double-stranded circular DNA molecule by a Cas nuclease, such as Cas9, within the cells can lead to the degradation of the desired linear DNA product, as the free ends of linear DNA are recognized by corresponding DNases. Furthermore, the producing cell itself can also be damaged. To mitigate this effect, it is preferable to use a regulatable promoter for the expression of type II Cas nucleases, such as Cas9, that exhibits the lowest possible basal expression.

[0073] For the inducible expression of Cas nucleases, such as Cas9, limiting promoter derepression, i.e., the inducible expression level, is also advantageous. Excessive expression of Cas nucleases, such as Cas9, over a short period can have similarly negative effects on pDNA quality and the producing cell as high basal expression over a longer period. Therefore, promoters exhibiting low basal expression and moderate expression levels after induction are particularly desirable.

[0074] Inducible expression of the guide RNA has the advantage over constitutive expression that the expression does not place a constant burden on the producing cell. Due to the reduced stress, the cell can produce more of the actual target product, the circular DNA.

[0075] By using a regulatable or inducible promoter, the timing of Cas9 nuclease and guide RNA production can be precisely controlled through expression induction. Regulatable or inducible expression of the processing machinery—that is, Cas9 nuclease and guide RNA—within the cell allows for the elimination of individual steps in the subsequent process, leading to economic advantages. For example, additional linearization steps or separate purification of the plasmid DNA can be omitted.

[0076] The method according to the invention is preferably characterized in that the expression of the Cas nuclease type II and / or the guide RNA is inducible. This means that the nucleotide sequence encoding the guide RNA and / or the nucleotide sequence encoding the Cas nuclease is operatively linked to an inducible promoter. Preferably, both the nucleotide sequence encoding the guide RNA and the nucleotide sequence encoding the Cas nuclease are operatively linked to an inducible promoter.

[0077] In one embodiment, the nucleotide sequence encoding the guide RNA and the nucleotide sequence encoding the Cas nuclease are operatively linked to the same inducible promoter. Using the same or a similar induction system, the expression of the guide RNA and the Cas nuclease begins simultaneously. This approach reduces cellular stress and, by synchronizing the onset of expression, allows for a more suitable ratio of Cas endonuclease to guide RNA molecules.

[0078] In another embodiment, the nucleotide sequence encoding the guide RNA and the nucleotide sequence encoding the Cas nuclease are operatively linked to different inducible promoters, e.g. for sequential expression.

[0079] Inducible promoters are known to those skilled in the art. Examples of suitable inducible promoters include the lac, tac, trc, lambda PL, ara, cumat, or tet promoters, or sequences derived from them.

[0080] According to the invention, a lactose-inducible promoter is preferably used as the inducible promoter, in particular the promoter WAC003 with the sequence SEQ ID NO:6 or the promoter WAC003-LacIDel with the sequence SEQ ID NO:7.

[0081] In one embodiment, the nucleotide sequence encodes the guide RNA and / or the nucleotide sequence encodes the Cas nuclease into the genome of E. coli integrated. In a preferred embodiment, both the nucleotide sequence encoding the guide RNA and the nucleotide sequence encoding the Cas nuclease are integrated into the genome of E . coli integrated. In another preferred embodiment, the nucleotide sequence encodes the Cas nuclease into the genome of E. coli The nucleotide sequence is integrated, but not the nucleotide sequence encoding the guide RNA. 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).

[0082] In another aspect, the present invention relates to the use of a Cas nuclease of type II for the linearization of a double-stranded, circular DNA molecule, wherein the linear DNA molecule on the template strand comprises in the 3' to 5' direction: (i) a template sequence surgically linked to an RNA polymerase promoter, (ii) a poly T sequence, (iii) a nucleotide sequence represented by N 1 N 2 N 3, wherein N 1 to N 3 each independently represent G, T, A or C or a nucleotide analogue thereof, and (iv) a protospacer adjacent motif (PAM).

[0083] In another aspect, the present invention relates to the use of a recombinant microorganism, wherein the nucleotide sequence encoding a guide RNA and / or the nucleotide sequence encoding a Cas nuclease of type II is integrated into the genome, for the production of a linear double-stranded DNA molecule, wherein the linear DNA molecule has smooth ends, and wherein the linear DNA molecule has a poly T sequence at the 5' end of the template strand.

[0084] One embodiment relates to the use of a recombinant microorganism in which the nucleotide sequence encoding a guide RNA and the nucleotide sequence encoding a type II Cas nuclease are integrated into the genome. Another embodiment relates to the use of a recombinant microorganism in which the nucleotide sequence encoding a type II Cas nuclease is integrated into the genome for the production of a linear double-stranded DNA molecule.

[0085] The recombinant microorganism can be any microorganism suitable for producing plasmid DNA. The preferred recombinant microorganism is... E. coli, The recombinant microorganism is preferred. E. coli K12 strain, the recombinant microorganism is particularly preferred. E. coli NEB10Beta.

[0086] The invention will be described in more detail below with reference to exemplary embodiments and the accompanying illustrations, without being limited by this.

[0087] Abbildung 1 Figure 1 shows an agarose gel with gel electrophoretically separated plasmid DNA and various controls: 1: Marker (GeneRuler™ < 1 kb DNA Ladder) 2: pGFP1 without incubation (negative control 1) 3: pGFP1 in water with incubation (negative control 2) 4: pGFP1 in cutting buffer (negative control 3) 5: pGFP1 in cutting buffer + 1 mM EDTA (negative control 4) 6: pGFP1 incubated with XbaI (positive control 1) 7: pGFP1 incubated with NcoI (positive control 2) 8: pGFP1 incubated with 25 nM Cas9 (negative control 5) 9: pGFP1 incubated with 25 nM sgRNA (negative control 6) 10-15: Test samples 1-5 (1-fold, 2-fold, 3-fold, 4-fold, 5-fold and 10-fold excess of Cas9 / guide RNA to pDNA).

[0088] Abbildung 2 shows an agarose gel with gel electrophoretically separated mRNAs that are in in In vitro transcriptions were generated using different DNA templates. Cas9 refers to the sample in which DNA linearized by SpCas9 / guide RNA was used as template DNA. Type IIS refers to the sample in which DNA linearized by the Type IIS restriction enzyme was used as template DNA. PCR refers to the sample in which DNA generated by PCR was used as template DNA. pGFP and pGFP2 refer to the samples in which the circular plasmids pGFP1 and pGFP2 were used as DNA templates.

[0089] Abbildung 3 Figure 1 shows the analysis of the poly(A) appendage by acrylamide gel electrophoresis. 1: Marker, GeneRuler™ < 1 kb DNA Ladder (Thermo Fisher Scientific). Cas9: RT-PCR product for RNA produced from template DNA linearized by SpCas9. Type IIS: RT-PCR product for RNA produced from template DNA linearized by a Type IIS restriction enzyme. PCR: RT-PCR product for RNA produced from template DNA generated by PCR.

[0090] Abbildung 4 shows the basal expression and expression strength of the modified promoters WAC003 and WAC003-LacIDel compared to the pTac1 baseline variant, using the expression of the reporter protein GFP as an example. E.coli K12.

[0091] Abbildung 5 shows a schematic representation of the plasmid pCas9_tracrRNA_CRISPR.

[0092] Figure 6is a schematic representation of a construct for genomic integration using λ-Red recombination. Examples Example 1: Plasmid DNA cutting activity by Cas9

[0093] First, a construct was designed for the production of the plasmid, consisting of a template sequence, a poly A attachment, and a target sequence for the cleavage of a Cas nuclease and PAM. This construct encodes a GFP mRNA template sequence and, at the 3' end of this sequence, a poly A attachment, which in turn is separated by three further nucleotides (5'-GTC-3') and the Protospacer Adjacent Motif(PAM) 5'-CGG-3' is followed. The PAM sequence represents the Cas9 gRNA recognition sequence for processing. To generate the template DNA sequence, the gfp gene (Uniprot: C5MKY7) was amplified with the two corresponding primers (SEQ ID NO: 8 and 9) in a PCR according to a standard protocol using Phusion polymerase (Thermo Fisher Scientific). The two primers comprised, on the one hand, the T7 RNA polymerase promoter (forward primer, SEQ ID NO: 8) and, on the other hand, the elements: Poly T, 5' GAC 3' and PAM (5'-CGG-3') (reverse primer, SEQ ID NO: 9). The PCR product (SEQ ID NO: 10) was then processed via the XbaI- and EcoRI site sites were cloned into a pUC18 vector (New England Biolabs). The resulting plasmid pGFP1 (SEQ ID NO:11) was used as the starting plasmid for the production of linearized template DNA in the following experiments / examples.

[0094] After successful production of the pGFP1 plasmid, the plasmid DNA was synthesized using E. coliNEB10Beta (New England Biolabs) was produced. For this purpose, the NEB10Beta cells were transformed with the pGFP1 plasmid using a heat shock method according to the manufacturer's instructions. The cells and 100 ng of pGFP1 were incubated on ice for 30 min before a 30-second heat shock at 42 °C. After a regeneration phase (1 h at 37 °C), the transformants were selected on selection medium (LB ampicillin (LB-Amp)). The resulting clones were grown in 3 mL cultures under selective conditions, and the plasmid of the clones was verified by restriction patterns and sequencing. Clones with the desired plasmid were grown in 50 mL of LB-Amp (shaking, 37 °C overnight) to generate sufficient amounts of plasmid DNA. Depending on the clone, an OD 600 (optical density at 600nm against water) of 8-14 was achieved in the shake flask.Finally, the plasmid DNA was isolated and purified from the overnight culture (stationary phase was reached) using the Gene Jet Plasmid Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0095] In the next step, a template DNA was created for the in vitro The synthesis of a guide RNA (SEQ ID NO:1) was designed. To enable the synthesis of a guide RNA with a specific length and sequence, the DNA sequence complementary to the guide RNA 3' was inserted downstream of a T7 RNA polymerase promoter sequence (SEQ ID NO:12). Together with the complementary sequence of the T7 promoter (SEQ ID NO:13), this was synthesized as a long single strand (primer) (Metabion).

[0096] To produce the guide RNA (SEQ ID NO:1), these two DNA oligonucleotides (SEQ ID NO:12 and 13) were hybridized by mixing them in equimolar amounts (100 pmol), boiling them at 95 °C for 5 minutes, and then slowly cooling them to room temperature. The resulting hybrid was then used as a template to synthesize the guide RNA with a in The RNA was synthesized using a vitro transcription kit (HighYield T7 RNA Synthesis Kit from Jena Bioscience) according to the manufacturer's instructions. The transcription reaction was then purified using the Monarch RNA Cleanup Kit from New England Biolabs (NEB) according to the manufacturer's instructions, and the concentration was determined by UV absorption.

[0097] After preparation of the pGFP1 plasmid (SEQ ID NO:11) and the guide RNA (SEQ ID NO:1), both were incubated together with the Cas nuclease SpCas9 (New England Biolabs) in a cutting buffer (20 mM Hepes pH 7.5; 150 mM KCl, 10 mM MgCl₂, 0.5 mM DTT, 0.1 mM EDTA). SpCas9 and the guide RNA were used in equimolar amounts and in varying excesses relative to the pDNA (5 nM) (1-fold, 2-fold, 3-fold, 4-fold, 5-fold, and 10-fold). To improve complex formation, SpCas9 and the guide RNA were first incubated separately (15 min, 37 °C). Subsequently, the pDNA (5 nM) and the reaction buffer were added. After 60 min incubation at 37 °C, the reaction was stopped by adding 20 µL of 2x formamide buffer (Thermo Fisher Scientific) and the pDNA was analyzed by agarose gel electrophoresis ( Figure 1 ).

[0098] For analysis, the cut pDNA was separated over a 1% agarose gel (TAE buffered) and subsequently visualized using Midori Green (Nippon Genetics Europe) on a gel documentation system (BioRad) (see Figure 1 The following served as controls: uncut and unincubated plasmid DNA, uncut incubated (1h, 37 °C) pDNA, pDNA with cutting buffer, pDNA with cutting buffer and 1 mM EDTA, pDNA with 25 nM Cas9 without guide RNA, pDNA with 25 nM guide RNA without Cas9, and as a positive control pDNA (SEQ ID NO:11), which was prepared using XbaI or NcoI (used according to manufacturer's specifications, Thermo Fisher Scientific) was linearized.

[0099] Figure 1This shows that the plasmid DNA pGFP1 is linearized by the Cas9 / guide RNA complex. The marker GeneRuler™ < 1 kb DNA Ladder (Thermo Fisher Scientific) was plotted in lane 1. The following served as controls: pGFP1 without incubation (lane 2, starting material), pGFP1 in water with incubation (lane 3, without Cas9 and guide RNA), pGFP1 in cutting buffer (lane 4, without Cas9 and guide RNA), pGFP1 in cutting buffer + 1 mM EDTA (lane 5, without Cas9 and guide RNA), pGFP1 incubated with XbaI (lane 6), pGFP1 incubated with NcoI (lane 7), pGFP1 incubated with 25 nM Cas9 (without guide RNA; lane 8), pGFP1 incubated with 25 nM guide RNA (without Cas9; lane 9). The test samples were applied in lanes 10-15 (1x, 2x, 3x, 4x, 5x and 10x excess Cas9 / guide RNA to pDNA). Figure 1Furthermore, it shows that with increasing amounts of the Cas9 / guide RNA complex, more pDNA is linearized. At a 10-fold molar excess (lane 15), almost the same degree of linearization is achieved as with conventional type II restriction enzymes. This is exemplified by the reactions using XbaI and NcoI (lanes 6 & 7). The dependence on Cas9 / guide RNA complex formation is shown in lanes 8 and 9, in which, with a 10-fold molar excess, one component—either the guide RNA (lane 8) or Cas9 (lane 9)—was omitted. No linearization is observed here, just as in pGFP1 samples in which no enzymes were used (lane 3, lane 4, lane 5). Example 2: mRNA synthesis from pDNA cut by Cas9

[0100] The plasmid DNA pGFP1 (SEQ ID NO:11) was linearized as described in Example 1 using SpCas9 and the guide RNA (SEQ ID NO:1) (10-fold molar excess, see also Figure 1 , Lane 15). The linearized plasmid was then purified using the Gene Jet PCR Purification Kit (Thermo Fisher Scientific). The linear, purified pDNA was then used as template DNA in a in A vitro transcription reaction (HighYield T7 RNA Synthesis Kit, Jena Bioscience) was used according to the manufacturer's instructions to synthesize mRNA. The reaction was carried out for 2 h at 37 °C using 200 ng of template DNA. To remove impurities and kit components, such as enzymes and nucleotides, the mRNA was purified using a Monarch RNA Cleanup Kit (NEB) according to the manufacturer's instructions.

[0101] To compare mRNA production processes using the method according to the invention with prior art methods, i.e., linearization of a plasmid DNA using type IIS restriction enzymes or production of a PCR product as linear starting DNA, pGFP2 (SEQ ID NO:14, produced analogously to pGFP1 by amplification of the gfp gene using primers of SEQ ID NO:9 and 15) was treated with the type IIS restriction enzyme. BpiIThe DNA was treated and linearized according to the manufacturer's instructions (Thermo Fisher Scientific). Additionally, a linear starting DNA was generated via a polymerase chain reaction (PCR product) (SEQ ID NO:16). This involved using a primer with a T7 RNA polymerase promoter (forward primer, SEQ ID NO:17) together with a primer containing a 60 nt poly(A) attachment (reverse primer, SEQ ID NO:18), as well as pGFP1 as the PCR template (10 ng) and Phusion DNA polymerase. The reaction was performed in the supplied buffer system according to the manufacturer's instructions (Thermo Fisher Scientific). The PCR product was then treated with DpnI Digested to remove any remaining methylated plasmid DNA from the PCR reaction. Finally, both the PCR product and the type IIS linearized plasmid were purified using a PCR Purification Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. While the linear DNA from the PCR reaction and the DNA linearized with SpCas9 were both smooth (" blunt ") ends, the type IIS linearized DNA has a 3' overhang.

[0102] The linear DNAs from the restriction digest and PCR reaction were added in the same quantities as the pDNA linearized using SpCas9 (200 ng) according to the manufacturer's instructions in a in vitro A transcription reaction was performed using template DNA for the synthesis of mRNA (HighYield T7 RNA Synthesis Kit, Jena Bioscience). Synthesis was carried out at 37°C for 2 hours. Finally, the mRNAs were purified using the Monarch RNA Cleanup Kit (NEB) according to the manufacturer's instructions.

[0103] The analysis of the synthesized mRNAs was initially performed via polyacrylamide gel electrophoresis. 200 ng of mRNA from each batch was mixed with RNA loading buffer (Gel Loading Buffer 2, Thermo Fisher Scientific) and boiled at 95 °C for 5 minutes. The RNA species were then analyzed by separation on a 15% TBE-UREA polyacrylamide gel (Thermo Fisher Scientific; 250 V, 50 min). Following electrophoresis, the gel was stained using SYBR Green II RNA Gel Stain (Thermo Fisher Scientific) and visualized in a gel documentation system (BioRad). Figure 2 The following served as controls: in vitro Transcription assays were performed using 200 ng of uncut pGFP1 and pGFP2. pGFP2 is a pGFP1 variant that lacks the PAM sequence (SEQ ID NO:14). The concentration of the generated mRNAs was determined photometrically using a NanoDrop photospectrometer (Thermo Fisher Scientific).

[0104] Figure 2This is a visualization of the produced mRNAs. Lane 1 contains the Century Marker™ (Thermo Fisher Scientific), and the following lanes contain the RNA samples corresponding to the starting DNAs: SpCas9 / guide RNA linearized DNA (Cas9), DNA linearized with type IIS restriction enzyme (Type IIS), DNA from the PCR reaction (PCR), and the uncut plasmids pGFP1 and pGFP2. Figure 2 This clearly shows that the main mRNA products obtained from reactions with linearized plasmids (Cas9, type IIS) and PCR product are identical in size. Only reactions with non-linearized plasmids (pGFP1 and pGFP2) yield larger products. These products result from the "unstopped" in In vitro transcription of T7 RNA polymerase. Due to the lack of linearization, no "Run-OFF" The polymerase can cause a "read-through" effect, resulting in heterogeneous, larger products.

[0105] For the analysis of the poly(A) attachment of mRNAs, an adapter oligonucleotide (SEQ ID NO:19) was ligated to the 3' end of the mRNA using T4 RNA ligase 1 (NEB). T4 RNA ligase 1 is an enzyme capable of ligating single-stranded RNA. In this process, a 5' monophosphate-containing substrate is ligated to the 3' OH end of the recipient mRNA. The 3' OH end naturally contains the synthesized RNA. The adapter oligonucleotide was synthesized to contain a 5' monophosphate group (metabion). To prevent self-ligation of the adapter oligonucleotide, the 3' end of the adapter oligonucleotide was also modified with a synthetic phosphate group. For ligation, 5 µg of RNA and 40 pmol of the adapter oligonucleotide were used. The reaction (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 for 2 h at 25°C. A buffer change to water was then performed using an amicon (Merck) (100 kDa cut-off).

[0106] The resulting adapter ligation products (adapter oligonucleotide + mRNA) were subsequently amplified by reverse transcriptase (RT-PCR). The SuperScript™ kit (Thermo Fisher Scientific) was used for the RT-PCR according to the manufacturer's instructions. Specific primers were used that bind directly 5' upstream of poly A (forward, SEQ ID NO:20) and to the adapter sequence (reverse, SEQ ID NO:21). The RT-PCR products were purified using a Thermo Fisher PCR Purification Kit according to the manufacturer's instructions, and the samples were then analyzed by electrophoresis in a 15% polyacrylamide gel. The separated DNA was stained with Midori Green (Biozym) and visualized in a gel documentation system (BioRad) (see [reference]). Figure 3 ).

[0107] Figure 3This shows the analysis of the poly A appendage by acrylamide gel electrophoresis. In lane 1, the marker GeneRuler™ < 1 kb DNA Ladder (Thermo Fisher Scientific) was applied. In the following 3 lanes, the RT-PCR products for the RNAs, which were prepared with differently linearized DNA templates, were separated (SpCas9 linearized (lane 2), linearized with type IIS restriction enzyme (lane 3), and PCR product (lane 4)).

[0108] In Figure 3 It can be seen that the same products with the exact same size distribution of the RT-PCR products were obtained in all 3 RNA samples. Since the source RNAs already show very high similarities (cf. Figure 2It can be assumed that the RT-PCR used the same or very similar templates as starting material. The gel patterns thus show that the method according to the invention not only effectively linearizes pDNA, but also linearizes the pDNA at the correct, predefined location, resulting in the desired unmasked free poly A attachment. Example 3: Promoter design for an inducible expression of Cas9

[0109] Since constitutive expression of a Cas endonuclease, for example Cas9, in a bacterial cell in vivo Since nonspecific DNA cutting events can occur at various locations in both plasmids and the chromosome of a bacterial cell, and thus damage the bacterial cell as a plasmid producer, inducible expression of the Cas endonuclease, for example Cas9, under a regulatable promoter with the lowest possible basal expression is preferred.

[0110] For the inducible expression of the Cas nuclease and thus the ability to "switch on" at a desired cultivation time via inducer addition in vivo Linearization of the plasmid product also favors limiting the inducible expression level. Excessive expression of a Cas nuclease over a short period can have similarly negative effects on the plasmid DNA and the producer cell as high basal expression over a longer period. Therefore, promoters that exhibit both low basal expression and moderate expression levels after induction are particularly advantageous.

[0111] The pTac1 promoter (de Boehr, 1983, Proceedings of the National Academy of Sciences 80(1):21-5), a hybrid of trp and lac promoters, retains only one operator site, LacO1, from the original Lac operon at position +1 (transcription start), for binding the repressor protein LacI. The repressor protein LacI itself, consisting of an N-terminal headpiece DNA-binding unit, a hinge region, a core region with N-terminal and C-terminal subdomains including a lactose-binding site and dimerization motif, and a C-terminal mini-zipper for tetramerization, associates to form a dimer of dimers. Each LacI dimer binds a 21 bp duplex DNA operator sequence, LacO (Lewis 2011. J Mol Biol. 409, 14-27). Two duplex DNA operator sites (LacO1 and LacO3) are simultaneously bound to the associated LacI tetramer in the native Lac operon, forming a so-called lac operon in the DNA strand. "repression loop"for maximum repression (Oehler et al. 2006, Nucleic Acids Res. 34, 606-612).

[0112] In WAC003 (SEQ ID NO:6) and WAC003-LacIdel (SEQ ID NO:7), a synthetic LacO binding site was inserted via the -35 region of the pTac1 promoter without altering the nucleotide sequences of the -35 and -10 promoter regions, the spacer between the two regions, or the nucleotide sequence of the spacer itself. WAC003 interacts with the native, i.e., tetramerizable, LacI repressor protein. In the WAC003-LacIdel construct, the gene lac I is replaced by a variant LacIdel which, as a result of a nucleotide frameshift at the C-terminus of the encoded LacI protein, forms a different amino acid sequence and thus can no longer form the LacI tetramer, but only the LacI dimer.

[0113] Basal expression (repression) and the expression achievable after inducer addition (derepression) were assessed using the reporter protein GFP. GFP, as a cytoplasmically localized protein, does not itself pose a burden to the cells and is therefore well-suited as a reference protein for measuring expression levels. The gene for GFP was placed under the control of the WAC003 and WAC003-LacIdel promoter constructs on a plasmid containing ColE1. origin of replication cloned, the plasmids into a E. coli K12 strain was transformed and the basal expression as well as the inducible expression were tested in shake culture in LB medium as follows.

[0114] Main cultures were inoculated from pre-cultures at OD 600 = 0.1. After 2 h, at a correlating cell density of OD 600 ~0.4, no basal expression was applied, or 0.1 mM IPTG was added (induction). After a further 24 h of cultivation, the amount of expressed GFP was measured (excitation 380 nm, emission 500 nm) and normalized to the respective underlying cell density OD 600 of the culture. A cell line with the pTac1 promoter (high basal expression, high inducible expression) was included as a control.

[0115] Figure 4This figure shows the basal expression and expression level of the WAC003 and WAC003-LacIdel promoter constructs compared to the Tac1 baseline. The WAC003 variant showed a significantly reduced basal expression compared to Tac1. After IPTG addition, the derepression of the WAC003 promoter was also significantly reduced (only ~16% GFP / OD 600 compared to Tac1). In the WAC003-LacIdel construct, the basal expression is also low; the inducible expression level is somewhat higher than in WAC003, but still very low compared to Tac1. The WAC003 and WAC003-LacIdel promoters are therefore very well suited for the inducible expression of a Cas nuclease, for example, Cas9, which in vivo is intended to be expressed for the specific linearization of plasmid DNA. Example 4: In vivo induction of Cas9 and purification of linear DNA after incubation

[0116] To analyze the inTo demonstrate the vivo functionality of the system described in Example 1 and Example 2, both the gene encoding SpCas9 (SEQ ID NO:2) and the construct for the guide RNA (SEQ ID NO:1) were cloned in the same plasmid (pWAC003, SEQ ID NO:5) under the inducible promoter WAC003-LacIDel (SEQ ID NO:7). Two SpCas9-specific primers (SEQ ID NO:22 and 23) were used to... Sp_cas9- The gene for cloning was amplified from the plasmid pCas9-ts1 (SEQ ID NO:24). The resulting PCR product was then analyzed via Eco RI and XbaI The plasmid pWAC003 (SEQ ID NO:5) was cloned downstream of the promoter WAC003-LacIDel (SEQ ID NO:7). The Cas9 expression cassette of the resulting plasmid pCas9 (SEQ ID NO:25) was sequenced using specific primers (SEQ ID NO:26 and SEQ ID NO:27) (Sequiserve) to confirm the desired sequence.

[0117] Since in a inSince the production of the guide RNA in a vivo system cannot be carried out via run-off transcription, as described in Example 2, the RNA design must be adapted. To generate a functional guide RNA, the natural form of RNA processing used in CRISPR-Cas9 systems must be employed. This means that a minimal CRISPR array and a tracrRNA are provided.

[0118] A tracrRNA sequence (SEQ ID NO:28) and a CRISPR sequence (SEQ ID NO:29) were inserted into the plasmid pCas9 (SEQ ID NO:25). In addition to the tracrRNA sequence and the CRISPR sequence, respectively, the sequences SEQ ID NO:28 and SEQ ID NO:29 each comprise a WAC003-LacIdel promoter sequence and a Rho-independent terminator sequence into which the tracrRNA or CRISPR sequence is embedded (5'-3'). For this purpose, the two DNA fragments were first prepared by PCR. Primers with SEQ ID NO:30-35 were used for SEQ ID NO:28, and primers with SEQ ID NO:36-41 were used for SEQ ID NO:29. The tracrRNA sequence was then modified using the two restriction enzymes KpnI and XbaI The pCas9 plasmid was cloned so that it is located 3' downstream of the Cas9 construct. The resulting plasmid, pCas9_tracrRNA (SEQ ID NO:42), was then populated with the CRISPR sequence via the cleavage sites. Christmas I and XbaIThe 3' position downstream of the tracrRNA sequence was cloned. The correct assembly of the resulting plasmid pCas9_tracrRNA_CRISPR (SEQ ID NO:43) was verified by sequencing using the specific primer (SEQ ID NO:27).

[0119] Figure 5 Figure 1 shows a schematic representation of the plasmid pCas9_tracrRNA_CRISPR. In the plasmid pCas9_tracrRNA_CRISPR, all three elements Cas9, tracrRNA and CRISPR are each under the control of a WAC003-LacIdel promoter and additionally possess a Rho-independent terminator sequence.

[0120] The plasmid pCas9_tracrRNA_CRISPR (SEQ ID NO:43) was developed together with pGFP1 (SEQ ID NO:11) in E. coliNEB10β was transformed. After selection for LB ampicillin / tetracycline, clones were inoculated in 20 mL of LB medium with antibiotics (100 mg / L ampicillin or 20 mg / L tetracycline) and incubated overnight at 37 °C (200 rpm). 100 mL of fresh LB medium was inoculated with ampicillin or tetracycline (100 mg / L or 20 mg / L, respectively) from these overnight cultures. The resulting main culture was incubated at 37 °C with shaking until an OD of 0.8 (600 nm) was reached. Upon reaching the target OD, IPTG was added to a final concentration of 0.5 mM to simultaneously induce expression of Cas9, the CRISPR cassette, and tracrRNA. Due to the co-expression of these elements, the pGFP1 plasmid is specifically cleaved. Two hours after induction, the cells were sedimented by centrifugation. The DNA in the supernatant (linearized pGFP1 and circular pCas9_tracrRNA_crRNA) was then isolated and purified according to the manufacturer's instructions using the Gene Jet Plasmid Kit (Thermo Fisher Scientific).

[0121] The composition of the isolated DNA and the proportion of linearized pGFP1 were analyzed using agarose gel electrophoresis and capillary electrophoresis. For agarose gel electrophoresis, the isolated DNA was separated over a 1.5% agarose gel and subsequently visualized using a Midori Green (Nippon Genetics Europe) and a gel documentation system (BioRad).

[0122] The usability of linearized DNA as a template for mRNA production was demonstrated as described in Example 2 via a in Vitro transcription was verified. The obtained RNAs were analyzed by polyacrylamide gel electrophoresis, and the poly A attachments of the mRNAs were analyzed using the described method with T4 RNA ligase 1 RT-PCR. Example 5: Incorporation of the cas9-CRISPR-tracrRNA construct onto the chromosome of a selected E. coli Tribe

[0123] The CRISPR-Cas construct pCas9_tracrRNA_CRISPR (SEQ ID NO:43) described in Example 4 was inserted into the chromosome of E. coliNEB10Beta was integrated. For the integration, an intergenic region of the gene locus of atpI The gene deletion method using λ-Red recombination according to Datsenko and Wanner (Datsenko et al. 2000, Proc. Natl. Acad. Sci. US A. 97: 6640 - 5) was chosen for the integration of the Cas9-gRNA construct.

[0124] For the integration of the Cas9-CRISPR-tracrRNA construct into the intergenic region of the atpI locus in the genome of the used E. coli The construct to be integrated was first designed using the NEB10Beta strain. In addition to the cas9_tracrRNA_CRISPR construct (SEQ ID NO:43) described in Example 4, this contains a chloramphenicol resistance cassette (cat) and two FRTs. sites, which flank the cat cassette, as well as a flanking homologous region to the integration site. The construct (SEQ ID NO:44) for genomic integration using λ-RED recombination is schematically shown in Figure 6The construct is shown as follows: Two homologous regions, each 150 bp long, flank the construct at the 3' and 5' ends. Between them, in a 5'-3' orientation, are the... cas9 _tracrRNA_CRISPR construct, an FRT site, the cat cassette and another FRT site.

[0125] The cat cassette is used for later selection to identify successful integration. The FRT sites are used later to recombine the cat to remove the cassette from the genome. The homologous regions flanking the entire construct are 150 base pairs long and serve for integration at the target site. The integration of the construct into the genome occurs through homologous recombination.

[0126] The complete construct was synthesized by GeneArt and cloned into the pMX vector (GeneArt). The construct was then excised from this plasmid using restriction enzymes (XhoI) and purified according to the manufacturer's instructions using a GeneJet gel extraction kit (Thermo Fisher Scientific).

[0127] To mediate homologous recombination for integration, the Lambda Red system, encoded on the plasmid pKD46 (CGSC: 7736), was used. The plasmid was transformed into NEB10Beta cells, and competent cells were then generated according to the procedure 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 resistance cassette was then performed. (catThe integration of the Cas9 or guide RNA construct into the chromosome (chloramphenicol acetyltransferase) was performed on LB agar plates containing 20 mg / L chloramphenicol. Verification of integration at the desired position in the chromosome was achieved using PCR with the oligonucleotides Check Fwd (SEQ ID NO:54) and Check Rev (SEQ ID NO:55) and chromosomal DNA from chloramphenicol-resistant cells as a template. As a negative control, PCR was also performed with chromosomal DNA from the original strain. E. coli NEB10Beta was performed. In this way, E . coli cells in which the Cas9 or guide RNA construct is integrated into the genome.

[0128] Subsequently, the cells were cured of the plasmid pKD46 according to the procedure described by Datsenko and Wanner (so), and the strain produced in this way was used as E. coli NEB10Beta atpI :: cas9 -CRISPR-tracrRNA- catThe removal of the chloramphenicol resistance cassette from the chromosome was performed according to the procedure of Datsenko and Wanner (so) using the plasmid pCP20 (CGSC: 7629), which encodes the FLP recombinase gene. The resulting strain, containing chloramphenicol-sensitive Cas9-CRISPR-tracrRNA, was then obtained using this method. E. coli NEB10Beta atpI :: cas9 The construct is designated as -CRISPR-tracrRNA. Successful integration of the construct (SEQ ID NO:44) was finally verified by sequencing using various primers (SEQ ID NO:45-54).

[0129] Into the new tribe (E. coliThe plasmid pGFP1 (SEQ ID NO:11) was then transformed into NEB10Beta atpI (cas9-CRISPR-tracrRNA) to test the system. After regeneration and selection (overnight on LB ampicillin agar plates), a colony was selected and used to start an overnight culture in 20 mL of LB ampicillin agar (37 °C, 200 rpm). This preculture was used to inoculate the 100 mL main culture. The resulting main culture was incubated until an OD of 0.8 (600 nm) was reached. Upon reaching the target OD, IPTG was added to initiate the expression of both Cas9 and the guide RNA. After 2 h under inducing conditions, the culture was harvested by centrifugation. The plasmid DNA in the cells was then isolated and purified according to the manufacturer's instructions using the Gene Jet Plasmid Kit (Thermo Fisher Scientific).

[0130] 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 properties (linearized, coiled) using a 1.5% agarose gel and subsequently visualized on a gel documentation system (BioRad) using Midori Green (Nippon Genetics Europe). The suitability of the DNA for mRNA synthesis was determined as described in Example 2. in Vitro transcription was tested.

Claims

1. Method for producing a linear double-stranded DNA molecule, the linear DNA molecule having blunt ends, and the linear DNA molecule having a poly T sequence at the 5' end of the template strand, comprising the steps of: (1) providing a double-stranded circular DNA molecule comprising on the template strand in the 3' to 5' direction: (i) a template sequence operatively 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, and (iv) a protospacer adjacent motif (PAM), (2) providing a guide RNA comprising a region which is complementary to the 5' end of the poly T sequence (ii) and to the nucleotide sequence (iii) and a region which interacts with a Cas nuclease, (3) providing a type II Cas nuclease, (4) contacting the double-stranded circular DNA molecule with the guide RNA and the CAS nuclease.

2. Method according to Claim 1, wherein the poly T sequence (ii) consists of between about 40 and about 250 T nucleotides, preferably between about 100 and about 140 T nucleotides, and particularly preferably about 120 T nucleotides.

3. Method according to Claim 1 or 2, wherein the region of the guide RNA which is complementary to the 5' end of the poly T sequence (ii) and to the nucleotide sequence (iii) has the sequence 5'-G(A)nN4N5N6-3', where n is a number between 17 and 23, where N4 to N6 are each independently G, U, A or C, and where the nucleotides N4N5N6 are complementary to the nucleotides N1N2N3 from (iii) .

4. Method according to any of Claims 1 to 3, wherein the type II Cas nuclease is Cas9, preferably selected from Cas9 from Streptococcus pyogenes having SEQ ID NO:2 or a sequence at least 70% homologous thereto, Cas9 from Streptococcus aureus having SEQ ID NO:3 or a sequence at least 70% homologous thereto and Cas9 from Streptococcus thermophilus having SEQ ID NO:4 or a sequence at least 70% homologous thereto.

5. Method according to any of Claims 1 to 4, wherein the type II Cas nuclease is Cas9 from Streptococcus pyogenes having SEQ ID NO:2 or a sequence at least 70% homologous thereto.

6. Method according to any of Claims 1 to 5, wherein the PAM has the sequence 5'-NGG-3', where N is independently selected from G, C, A and T.

7. Method according to any of Claims 1 to 6, wherein the poly T sequence (ii) consists of about 120 T nucleotides, wherein the region of the guide RNA which is complementary to the 5' end of the poly T sequence (ii) and to the nucleotide sequence (iii) has the sequence 5'-G(A)n - N4N5N6-3', where n is equal to 17, where N4 to N6 are each independently G, U or C, wherein the PAM has the sequence 5'-NGG-3', where N is independently selected from G, C, A and T, and wherein the type II Cas nuclease is Cas9 from Streptococcus pyogenes having SEQ ID NO:2 or a sequence at least 70% homologous thereto.

8. Method according to any of Claims 1 to 7, characterized in that the providing of the guide RNA is achieved by the expression of a nucleotide sequence encoding the guide RNA.

9. Method according to any of Claims 1 to 8, characterized in that the providing of the type II Cas nuclease is achieved by the expression of a nucleotide sequence encoding the Cas nuclease.

10. Method according to either of Claims 8 and 9, characterized in that the nucleotide sequence encoding the guide RNA and / or the nucleotide sequence encoding the Cas nuclease is / are operatively linked to an inducible promoter.

11. Method according to Claim 10, wherein the inducible promoter is a lactose-inducible promoter, in particular the promoter WAC003 having the sequence SEQ ID NO:6 or the promoter WAC003-LacDel having the sequence SEQ ID NO:7.

12. Method according to any of Claims 8-11, characterized in that the nucleotide sequence encoding the guide RNA and / or the nucleotide sequence encoding the Cas nuclease has / have been integrated into the genome of E. coli.

13. Use of a type II Cas nuclease for linearization of a double-stranded, circular DNA molecule, the linear DNA molecule comprising on the template strand in the 3' to 5' direction: (i) a template sequence operatively 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, and (iv) a protospacer adjacent motif (PAM).

14. Use of a recombinant microorganism in which the nucleotide sequence encoding a guide RNA and / or the nucleotide sequence encoding a type II Cas nuclease has / have been integrated into the genome for production of a linear double-stranded DNA molecule, the linear DNA molecule having blunt ends, and the linear DNA molecule having a poly T sequence at the 5' end of the template strand.

15. Use according to Claim 14, wherein the recombinant microorganism is E. coli, preferably an E. coli K12 strain, particularly preferably E. coli NEB10Beta.