WO2024146916A1

WO2024146916A1 - Activated bec nucleases for degrading nucleic acid molecules

Info

Publication number: WO2024146916A1
Application number: PCT/EP2024/050138
Authority: WO
Inventors: Paul Scholz; Michael Krohn; Rebecca HEGER
Original assignee: BRAIN Biotech AG
Priority date: 2023-01-04
Filing date: 2024-01-04
Publication date: 2024-07-11

Abstract

The present invention relates to methods and uses for degrading nucleic acid molecules in a sample by activating the nonspecific nuclease activity of a CRISPR nuclease having nonspecific nuclease activity. BEC family nucleases are able to degrade RNA, ssDNA and dsDNA in a collateral manner making them a group of versatile and robust nucleases in many applications.

Description

Activated BEC Nucleases for Degrading Nucleic Acid Molecules

The present invention relates to methods and uses for degrading nucleic acid molecules in a sample by activating the nonspecific nuclease activity of a CRISPR nuclease having nonspecific nuclease activity.

In this specification, a number of documents including patent applications and manufacturer’s manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

The genome editing technology CRISPR enables genome editing in a broad range of cells and organisms. The CRISPR-based technique substantially streamlined the targeted gene modification in different prokaryotic and eukaryotic cells.

The development of new CRISPR-Cas genome editing tools continues to drive major advances in the life sciences. As classes of CRISPR-Cas-derived genome editing agents- nucleases, base editors, transposases/recombinases and prime editors-are currently available for modifying genomes in experimental systems. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded their targeting scope and improved editing specificity (Anzalone at al. (2020) Nat Biotechnol 2020 Jul;38(7):824-844).

A further class of CRISPR-Cas-derived genome editing agents that broadens and will further broaden cell editing capabilities are the so-called BEC (BRAIN Engineered Cas) nucleases. The BEC nucleases are described in WO 2022/017633. BEC nucleases do not occur in nature but they are engineering CRISPR-Cas nucleases that were generated based on random mutagenesis and screening approaches. While it is described in WO 2022/017633 that BEC nucleases display a different molecular mechanism in comparison to classical CRISPR Cas nucleases, the exact molecular mechanism of BEC nucleases is yet to be fully understood. In a further step is then to be determined for what kind of applications BEC nucleases are useful based on their molecular mechanism.

The need to further elucidate the molecular mechanism of BEC nucleases and to identify on this basis novel applications of BEC nucleases is addressed by the present invention.

Hence, the present invention relates in first aspect to a method for degrading nucleic acid molecules in a sample, comprising (I) contacting the sample with (A) a ribonucleoprotein complex (RNP) comprising or consisting of (i) a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3; (b) an amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6 or 7; (c) an amino acid sequence being at least 80% identical to the amino acid sequence of (a), or (d) an amino acid sequence being encoded by a nucleic acid sequence being at least 80 % identical to the nucleotide sequence of (b), (ii) in complex with a guide RNA; and (B) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity (thereby degrading nucleic acid molecules in a sample); wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity; or (II) (a) activating the nonspecific nuclease activity of a CRISPR nuclease having nonspecific nuclease activity by contacting the RNP as defined in (I) with the activation RNA or the nucleic acid molecule encoding in expressible form the activation RNA as defined in (I) under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity, and (b) contacting the sample with the activated CRISPR nuclease having nonspecific nuclease activity (thereby degrading nucleic acid molecules in a sample).

The term “nucleic acid molecule” in accordance with the present invention includes DNA, such as cDNA or double or single stranded genomic DNA and RNA.

In this regard, "DNA" (deoxyribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide bases, that are linked together on a deoxyribose sugar backbone. DNA can have one strand of nucleotide bases, or two complementary strands which may form a double helix structure. "RNA" (ribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and uracil (II), called nucleotide bases, that are linked together on a ribose sugar backbone. RNA typically has one strand of nucleotide bases, such as mRNA. Included are also single- and double-stranded hybrids molecules, i.e. , DNA-DNA, DNA-RNA and RNA-RNA. The nucleic acid molecule is preferably dsDNA, ssDNA and/or ssRNA.

A nucleic acid molecule of the invention comprises with increasing preference at least 10, 15, 20, 25, 50, 75, 100, 250, 500 and 1000 nucleotide bases.

A nucleic acid molecule often carries genetic information, including the information used by cellular machinery to produce proteins and/or polypeptides. The nucleic acid molecule according to the invention may additionally comprise promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'-non-coding regions, and the like.

The nucleic acid molecule may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acid molecules, in the following also referred as polynucleotides, may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage.

Further included are nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers. Such nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2’-O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA) (see Braasch and Corey, Chem Biol 2001 , 8:1). LNA is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2’-oxygen and the 4’-carbon. Also included are nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil. Degradation of nucleic acid molecules as refer to herein includes that longer nucleic acid molecules are degraded into smaller fragments thereof. Hence, degradation nucleic acid molecules comprises fragmentation of nucleic acid molecules. This degradation is achieved by the activated CRISPR nuclease having nonspecific nuclease activity. The action and nature of this CRISPR nuclease will be further detailed herein below. The mean size of the degraded nucleic acid molecules is generally below 250 bps nucleotide bases.

Nucleases are enzymes cleaving the phosphodiester bonds of nucleic acids and may be, for example, CRISPR nucleases (or CRISPR-Cas nucleases or Cas nucleases), endo- or exonucleases, DNases or RNases, topoisomerases, recombinases, ribozymes, or RNA splicing enzymes. CRISPR nucleases are normally used for site-specific genome editing. For instance, the first CRISPR nuclease Cas9 together with synthetic guide RNA is a two-component system that introduces a targeted and site-specific double stranded break. This break activates repair through error prone non-homologous end joining (NHEJ) or Homology directed Repair (HDR). In the presence of a donor template with homology to the targeted locus, the HDR pathway operates allowing for precise mutations to be made.

In contrast, the CRISPR nuclease according to the present invention is a CRISPR nuclease that displays nonspecific nuclease activity upon its activation by an activation RNA together with a guide RNA (or gRNA). This may also be referred to a three-component system as compared to above-described two-component system for activating the site-specific double stranded break by classic CRISPR nucleases. Within the three-component system the guideRNA connects CRISPR nuclease and the activation RNA with the consequence that the CRISPR nuclease becomes activated and displays nonspecific nuclease activity. This nonspecific nuclease activity is also referred to herein as “collateral activity”.

The nonspecific nuclease activity means that the activated CRISPR nuclease of the invention cleaves the phosphodiester bonds of nucleic acid molecules (preferably dsDNA, ssDNA and/or ssRNA) in sample essentially without sequence specificity. This enables the degradation of all or essentially all nucleic acid molecules in a sample. To the best knowledge of the inventors such an activity of an activated CRISPR nuclease has not been reported in the prior art.

The CRISPR nuclease having nonspecific nuclease activity comprises or consists of (a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3; (b) an amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6 or 7; (c) an amino acid sequence being at least 80% identical to the amino acid sequence of (a), or (d) an amino acid sequence being encoded by a nucleic acid sequence being at least 80 % identical to the nucleotide sequence of (b).

The CRISPR nucleases to be used in accordance with the present invention are so-called BRAIN Engineered Cas proteins (BEC) nucleases. In more detail, the amino acid sequence of SEQ ID NO: 1 , 2 or 3 and the nucleotide sequence of SEQ ID NO: 4, 5 or 6 are the amino acid sequences and the nucleotide sequences of the class 2, type V RNA-guided DNA nucleases of BEC85, BEC67 and BEC10 as disclosed in the international application WO 2022/017633. Among the three BEC nucleases BEC10 is most preferred. SEQ ID NO: 7 is BEC10 that has been optimized for the expression in E. coli. SEQ ID NO: 7 is therefore also designated E.coliBECIO.

As mentioned, an activation-RNA and a guide RNA are required to activate the nonspecific nuclease activity of the CRISPR nuclease according to the invention.

The activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity.

The guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity.

It is to be understood that the activation RNA or a nucleic acid molecule encoding the activation RNA in expressible from and the guide RNA have been prepared as ex vivo or in vitro and that they do not occur in the sample (in the case of option (I) before they are added to the sample). It is also understood that the RNA encoded by the nucleic acid in expressible from is indeed expressed for the method of the invention to function.

The activation RNA mimics the genomic target locus that is usually targeted by a classic CRISPR nuclease, because it comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity. The PAM is a 2-6-base pair DNA sequence immediately following the first segment targeted by the CRISPR nuclease. The CRISPR nuclease only cleaves the first segment if it is followed by the PAM sequence. While the guide RNA in target-specific genome editing mediates the interaction between the CRISPR nucleases and the target locus to be genome edited, the guide RNA as used herein mediates the interaction between the CRISPR nucleases and the activation RNA. The guide RNA is a small piece of RNA with a short "guide" sequence that attaches (binds) to the activation RNA. Thus, the gRNA sequence is also called protospacer sequence herein and is preferably about 21 nucleotides long, wherein about is preferably ±20%, more preferably ±10%. Hence, the guide RNA brings the CRISPR nuclease to the activation RNA and then CRISPR nuclease becomes activated.

This initial and target-specific activation by the described interplay between CRISPR nuclease having nonspecific nuclease activity, the guide RNA and the activation RNA actives the CRISPR nuclease having nonspecific nuclease activity to be used in accordance with the invention. This initial and target-specific interplay of the CRISPR nuclease with the guide RNA and the activation RNA activates the nonspecific nuclease activity of the the CRISPR nuclease. The activated CRISPR nuclease can then degrade nucleic acid molecules within a sample. In this connection it is of note that the nonspecific nuclease activity is independent of the presence of the PAM sequence. Hence, the nucleic acid molecules in the sample are degraded by the nonspecific nuclease activity independently of the presence of the PAM sequence.

In accordance with option (I) herein the CRISPR nuclease having nonspecific nuclease activity is activated within the sample; i.e. the described interplay between CRISPR nuclease having nonspecific nuclease activity, the guide RNA and the activation RNA is within the sample comprising the nucleic acid molecules to be degraded. In accordance with option (II) herein the CRISPR nuclease having nonspecific nuclease activity is pre-activated; i.e. the described interplay between CRISPR nuclease having nonspecific nuclease activity, the guide RNA and the activation RNA is conducted in the absence of the sample comprising the nucleic acid molecules to be degraded and only then the sample is contacted with the already activated CRISPR nuclease.

In accordance with the first aspect of the invention, the gRNA and the CRISPR nuclease are complexed with each other and thereby form a ribonucleoprotein complex (RNP). Also in the appended examples such an RNP is used. RNPs are assembled in vitro and can be delivered to the cell by methods known in the art, for example, electroporation or lipofection. For introducing an RNP into a cell the same methods may be used as discussed for proteins herein below. RNPs are capable of cleaving the target site with comparable efficacy as nucleic acidbased (e.g. vector-based) CRISPR nucleases (Kim et al. (2014), Genome Research 24(6): 1012-1019). The activation RNA can be used as RNA or as a nucleic acid molecule (e.g. an expression vector) encoding in expressible form the activation RNA can be used.

The term “in expressible form” means that one or more nucleic acid molecules may encode their constituents in a form that ensures that an RNA (if being encoded) is transcribed and/or protein (if being encoded) is transcribed and translated in a sample or cell.

The nucleic acid molecule encoding in expressible form the activation RNA may be inserted into several commercially available expression vectors. Vector modification techniques are known in the art and, for example, described in Sambrook and Russel, 2001. Generally, vectors can contain one or more origins of replication (ori) and inheritance systems for cloning or expression, one or more markers for selection in the host, e.g., antibiotic resistance, and one or more expression cassettes. Suitable origins of replication include, for example, the Col E1 , the SV40 viral and the M 13 origins of replication. The nucleic acid sequences inserted in the vector can e.g. be synthesized by standard methods, or isolated from natural sources. Ligation of the coding sequences to transcriptional regulatory elements and/or to other amino acid encoding sequences can be carried out using established methods. Such regulatory sequences are well known to those skilled in the art and include, without being limiting, regulatory sequences ensuring the initiation of transcription, initiation of translation, internal ribosomal entry sites (IRES) or 2A linkers (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471 - 1476) and optionally regulatory elements ensuring termination of transcription and stabilization of the transcript. Non-limiting examples for regulatory elements (e.g. ensuring the initiation of transcription) comprise a translation initiation codon, enhancers such as e.g. the SV40- enhancer, insulators and/or promoters, such as for example the cytomegalovirus (CMV) promoter, elongation factor-1 alpha (EF1 -alpha), promoter, SV40-promoter, RSV-promoter (Rous sarcoma virus), the lacZ promoter, chicken beta-actin promoter, CAG-promoter (a combination of chicken beta-actin promoter and cytomegalovirus immediate-early enhancer), the gai10 promoter, human elongation factor 1a-promoter, AOX1 promoter, GAL1 promoter, CaM-kinase promoter, the lac, trp or tac promoter, the lacUV5 promoter, the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter or a globin intron in mammalian and other animal cells. Non-limiting examples for regulatory elements ensuring transcription termination include the V40-poly-A site, the tk-poly-A site, lacZ or AcMNPV polyhedral polyadenylation signals, which are to be included downstream of the nucleic acid sequence of the invention. Additional regulatory elements may include translational enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing, nucleotide sequences encoding secretion signals or, depending on the expression system used, signal sequences capable of directing the expressed polypeptide to a cellular compartment. Moreover, elements such as origin of replication, drug resistance gene, regulators (as part of an inducible promoter) may also be included.

Means for introducing proteins (or peptides) into living cells are known in the art and comprise but are not limited to microinjection, electroporation, lipofection (using liposomes), nanoparticle-based delivery, and protein transduction. Any one of these methods may be used for the introduction of the CRISPR nuclease into cells of a sample comprising nucleic acid molecules to be degraded.

A liposome used for lipofection is a small vesicle, composed of the same material as a cell membrane (i.e. , normally a lipid bilayer e.g. made of phospholipids), which can be filled with one or more protein(s) (e.g. Torchilin VP. (2006), Adv Drug Deliv Rev., 58(14):1532-55). To deliver a protein into a cell, the lipid bilayer of the liposome can fuse with the lipid bilayer of the cell membrane, thereby delivering the contained protein into the cell. It is preferred that the liposomes used in accordance with the invention are composed of cationic lipids. The cationic liposome strategy has been applied successfully to protein delivery (Zelphati et al. (2001). J. Biol. Chem. 276, 35103-35110). As known in the art, the exact composition and/or mixture of cationic lipids used can be altered, depending upon the protein(s) of interest and the cell type used (Feigner et al. (1994). J. Biol. Chem. 269, 2550-2561). Nanoparticle-based delivery of Cas9 ribonucleoprotein and donor DNA for the induction of homology-directed DNA repair is, for example, described in Lee et al. (2017), Nature Biomedical Engineering, 1 :889-90.

Protein transduction specifies the internalisation of proteins into the cell from the external environment (Ford et al (2001), Gene Therapy, 8:1 -4). This method relies on the inherent property of a small number of proteins and peptides (preferably 10 to 16 amino acids long) being able to penetrate the cell membrane. The transducing property of these molecules can be conferred upon proteins which are expressed as fusions with them and thus offer, for example, an alternative to gene therapy for the delivery of therapeutic proteins into target cells. Commonly used proteins or peptides being able to penetrate the cell membrane are, for example; the antennapedia peptide, the herpes simplex virus VP22 protein, HIV TAT protein transduction domain, peptides derived from neurotransmitters or hormones, or a 9xArg-tag.

Microinjection and electroporation are well known in the art and the skilled person knows how to perform these methods. Microinjection refers to the process of using a glass micropipette to introduce substances at a microscopic or borderline macroscopic level into a single living cell. Electroporation is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. By increasing permeability, protein (or peptides or nucleic acid sequences) can be introduced into the living cell.

Also means and methods for the introduction for the nucleic acid molecules as referred to herein into cells are known in the art and these methods encompass transducing or transfecting cells.

Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector. Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell's genome. Generally, a plasmid is constructed in which the genes to be transferred are flanked by viral sequences that are used by viral proteins to recognize and package the viral genome into viral particles. This plasmid is inserted (usually by transfection) into a producer cell together with other plasmids (DNA constructs) that carry the viral genes required for formation of infectious virions. In these producer cells, the viral proteins expressed by these packaging constructs bind the sequences on the DNA/RNA (depending on the type of viral vector) to be transferred and inserted into viral particles. For safety, none of the plasmids used contains all the sequences required for virus formation, so that simultaneous transfection of multiple plasmids is required to get infectious virions. Moreover, only the plasmid carrying the sequences to be transferred contains signals that allow the genetic materials to be packaged in virions, so that none of the genes encoding viral proteins are packaged. Viruses collected from these cells are then applied to the cells to be altered. The initial stages of these infections mimic infections with natural viruses and lead to expression of the genes transferred and (in the case of lentivirus/retrovirus vectors) insertion of the DNA to be transferred into the cellular genome. However, since the transferred genetic material does not encode any of the viral genes, these infections do not generate new viruses (the viruses are "replication-deficient").

T ransfection is the process of deliberately introducing naked or purified nucleic acids or purified proteins or assembled ribonucleoprotein complexes into cells. Transfection is generally a non- viral based method.

Transfection may be a chemical-based transfection. Chemical-based transfection can be divided into several kinds: transfection using cyclodextrin, polymers, liposomes, calcium phosphate or nanoparticles. One of the cheapest methods uses calcium phosphate. HEPES- buffered saline solution (HeBS) containing phosphate ions are combined with a calcium chloride solution containing the DNA to be transfected. When the two are combined, a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the DNA to be transfected on its surface. The suspension of the precipitate is then added to the cells to be transfected (usually a cell culture grown in a monolayer). By a process not entirely understood, the cells take up some of the precipitate, and with it, the DNA. This process has been a preferred method of identifying many oncogenes. Other methods use highly branched organic compounds, so-called dendrimers, to bind the DNA and transfer it into the cell. Another method is the use of cationic polymers such as DEAE-dextran or polyethylenimine (PEI). The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis. Lipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer. Lipofection generally uses a positively charged (cationic) lipid (cationic liposomes or mixtures) to form an aggregate with the negatively charged (anionic) genetic material. This transfection technology performs the same tasks in terms of transfer into cells as other biochemical procedures utilizing polymers, DEAE-dextran, calcium phosphate, and electroporation. The efficiency of lipofection can be improved by treating transfected cells with a mild heat shock. Fugene is a series of widely used proprietary non-liposomal transfection reagents capable of directly transfecting a wide variety of cells with high efficiency and low toxicity.

Transfection may also be a non-chemical method. Electroporation (gene electrotransfer) is a popular method, where transient increase in the permeability of cell membrane is achieved when the cells are exposed to short pulses of an intense electric field. Cell squeezing enables delivery of molecules into cells via cell membrane deformation. Sonoporation uses high- intensity ultrasound to induce pore formation in cell membranes. This pore formation is attributed mainly to the cavitation of gas bubbles interacting with nearby cell membranes since it is enhanced by the addition of ultrasound contrast agent, a source of cavitation nuclei. Optical transfection is a method where a tiny (~1 pm diameter) hole is transiently generated in the plasma membrane of a cell using a highly focused laser. Protoplast fusion is a technique in which transformed bacterial cells are treated with lysozyme in order to remove the cell wall. Following this, fusogenic agents (e.g., Sendai virus, PEG, electroporation) are used in order to fuse the protoplast carrying the gene of interest with the recipient target cell.

Finally, transfection may be a particle-based method. A direct approach to transfection is the gene gun, where the DNA is coupled to a nanoparticle of an inert solid (commonly gold), which is then "shot" (or particle bombardment) directly into the target cell's nucleus. Hence, the nucleic acid is delivered through membrane penetration at a high velocity, usually connected to microprojectiles. Magnetofection, or magnet-assisted transfection, is a transfection method that uses magnetic force to deliver DNA into target cells. Impalefection is carried out by impaling cells by elongated nanostructures and arrays of such nanostructures such as carbon nanofibers or silicon nanowires which have been functionalized with plasmid DNA.

It can be taken from the appended examples that it was surprisingly found that once a BEC- type CRISPR nuclease is activated by the initial target-specific activation it is capable of non- specifically degrading essentially all nucleic acid molecules in a sample. It is believed that all nucleic acid molecules (e.g. dsDNA, ssDNA and ssRNA) are degraded. This unexpected activity is used in appended Example 2 to deplete DNA and RNA from fermentation broth or supernatant thereby “cleaning” the fermentation broth from biologically active nucleic acid molecules. In Example 3 it is furthermore shown that BEC-type CRISPR nucleases exert rapid dsDNA collateral activity leading to the unspecific degradation of dsDNA. Unlike any other types of CRISPR nucleases BEC-type CRISPR nucleases are advantageously able to degrade RNA, ssDNA and dsDNA. As dsDNA is the most stable form of all nucleic acids it is the hardest to deplete from a fermentation broth and no other types of CRISPR nucleases are to the best knowledge of the inventors displaying a collateral activity targeting dsDNA. Therefore, BEC family nucleases feature an important technical advantage and a distinct mode of action in comparison to other types of CRISPR nucleases. Their ability to degrade dsDNA is unique. Biologically active nucleic acid molecules, in particular dsDNAs, are not only unwanted in fermentation broth but are unwanted in a number of various samples, such as industrial compositions, food or beverage compositions, cosmetic compositions, diagnostic compositions or pharmaceutical compositions. Hence, the method and uses of the present invention have broad applicability. To the best knowledge of the inventors BEC-type CRISPR nucleases are the first group of CRISPR nucleases that can be used to degrade essentially all nucleic acid molecules, including dsDNAs, in a sample and this activity of BEC-type CRISPR nucleases is described for the first time herein.

The present invention relates in a second aspect to a method for degrading nucleic acid molecules in a sample, comprising (I) contacting the sample with (A) one or more nucleic acid molecules, said one or more nucleic acid molecules encoding in expressible form (i) a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3; (b) an amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6 or 7; (c) an amino acid sequence being at least 80% identical to the amino acid sequence of (a), or (d) an amino acid sequence being encoded by a nucleic acid sequence being at least 80 % identical to the nucleotide sequence of (b), and (ii) a guide RNA; and (B) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity (thereby degrading nucleic acid molecules in a sample); wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity; or (II) (a) activating the nonspecific nuclease activity of a CRISPR nuclease having nonspecific nuclease activity by contacting the one or more nucleic acid molecules as defined in (I) with the activation RNA or the nucleic acid molecule encoding in expressible form the activation RNA as defined in (I) under conditions wherein the activation- RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity, and (b) contacting the sample with the activated CRISPR nuclease having nonspecific nuclease activity (thereby degrading nucleic acid molecules in a sample).

The present invention relates in a third aspect to a method for degrading nucleic acid molecules in a sample, comprising (I) contacting the sample with (A) a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3; (b) an amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6 or 7; (c) an amino acid sequence being at least 80% identical to the amino acid sequence of (a), or (d) an amino acid sequence being encoded by a nucleic acid sequence being at least 80 % identical to the nucleotide sequence of (b), and (B) a guide RNA; and (C) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity (thereby degrading nucleic acid molecules in a sample); wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity; or (II) (a) activating the nonspecific nuclease activity of a CRISPR nuclease having nonspecific nuclease activity by contacting the CRISPR nuclease having as defined in (I) with the guide RNA and the activation RNA or the nucleic acid molecule encoding in expressible form the activation RNA as defined in (I) under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity, and (b) contacting the sample with the activated CRISPR nuclease having nonspecific nuclease activity (thereby degrading nucleic acid molecules in a sample).

The present invention relates in a fourth aspect to a method for degrading nucleic acid molecules in a sample, comprising (I) contacting the sample with (A) a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3; (b) an amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6 or 7; (c) an amino acid sequence being at least 80% identical to the amino acid sequence of (a), or (d) an amino acid sequence being encoded by a nucleic acid sequence being at least 80 % identical to the nucleotide sequence of (b), and (B) a nucleic acid molecule encoding in expressible form a guide RNA; and (C) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity (thereby degrading nucleic acid molecules in a sample); wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity; or (II) (a) activating the nonspecific nuclease activity of a CRISPR nuclease having nonspecific nuclease activity by contacting the CRISPR nuclease having as defined in (I) with the nucleic acid molecule encoding in expressible form the guide RNA and the activation RNA or the nucleic acid molecule encoding in expressible form the activation RNA as defined in (I) under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity, and (b) contacting the sample with the activated CRISPR nuclease having nonspecific nuclease activity (thereby degrading nucleic acid molecules in a sample).

The present invention relates in a fifth aspect to a method for degrading nucleic acid molecules in a sample, comprising (I) contacting the sample with (A) a nucleic acid molecule encoding in expressible form a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3; (b) an amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6 or 7; (c) an amino acid sequence being at least 80% identical to the amino acid sequence of (a), or (d) an amino acid sequence being encoded by a nucleic acid sequence being at least 80 % identical to the nucleotide sequence of (b), and (B) a guide RNA; and (C) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity (thereby degrading nucleic acid molecules in a sample); wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity; or (II) (a) activating the nonspecific nuclease activity of a CRISPR nuclease having nonspecific nuclease activity by contacting the nucleic acid molecule encoding in expressible form the CRISPR nuclease having nonspecific nuclease activity as defined in (I) with the guide RNA and the activation RNA or the nucleic acid molecule encoding in expressible form the activation RNA as defined in (I) under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity, and (b) contacting the sample with the activated CRISPR nuclease having nonspecific nuclease activity (thereby degrading nucleic acid molecules in a sample).

The above definitions and preferred embodiments of the first aspect of the invention apply mutatis mutandis to the second to fifth aspect. The second to fifth aspect are related to the first aspect and all of the first to fifth aspect are related to different options how to finally arrive at conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity.

All aspects have in common that the activation RNA can be used as RNA or a nucleic acid molecule (e.g. an expression vector) encoding in expressible form the activation RNA can be used. Also all aspects have in common that in accordance with option (I) the CRISPR nuclease having nonspecific nuclease activity is activated within the sample while in accordance with option (II) the CRISPR nuclease having nonspecific nuclease activity is pre-activated and then added its active form to the sample.

The five aspects differ from each other regarding the form of the gRNA and the CRISPR nuclease being used.

As discussed above, in the first aspect the gRNA and the CRISPR nuclease are complexed with each other and thereby form a ribonucleoprotein complex (RNP). Hence, the gRNA is used as RNA, the CRISPR nuclease as protein and both together from the RNP. In the second aspect the one or more nucleic acid molecules are used that encode in expressible form the CRISPR nuclease having nonspecific nuclease activity and the guide RNA. Hence, neither the gRNA nor the CRISPR nuclease are “directly” used but instead one or more nucleic acid molecules encoding the same.

Single vectors containing both the CRISPR nuclease and the gRNAs or two separate vectors may be used. The use of an all-in-one vector that expresses the gRNA, the CRISPR nuclease and optionally also the activation RNA is preferred since only one vector is to be introduced into the cells. A vector which can express the CRISPR nuclease and up to seven gRNAs is, for example, described in Sakuma et al, Sci Rep. 2014; 4: 5400.

Many single gRNA empty vectors (with and without the CRISPR nuclease) are available in the art. Likewise several empty multiplex gRNA vectors are available that can be used to express multiple gRNAs from a single plasmid (with or without the expression of the CRISPR nuclease). Finally, also vectors are available that only express the CRISPR nuclease (see https://www.addgene.org/crispr/empty-grna-vectors/).

In the third aspect the gRNA is used as RNA and the CRISPR nuclease as protein - but in contrast to the first aspect - the gRNA and the CRISPR nuclease are not complexed together to from the RNP.

In the fourth aspect the the CRISPR nuclease is used as protein and a nucleic acid molecule encoding in expressible form a guide RNA.

Finally, in the fifth aspect a nucleic acid molecule encoding in expressible form the CRISPR nuclease is used and the guide RNA as RNA.

The present invention relates in a sixth aspect to the use of (A) a ribonucleoprotein complex (RNP) comprising or consisting of (i) a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3; (b) an amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6 or 7; (c) an amino acid sequence being at least 80% identical to the amino acid sequence of (a), or (d) an amino acid sequence being encoded by a nucleic acid sequence being at least 80 % identical to the nucleotide sequence of (b), (ii) in complex with a guide RNA; and (B) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA for degrading the nucleic acid molecules in a sample; wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity.

The present invention relates in a seventh aspect to the use of (A) one or more nucleic acid molecules, said one or more nucleic acid molecules encoding in expressible form (i) a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3; (b) an amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6 or 7; (c) an amino acid sequence being at least 80% identical to the amino acid sequence of (a), or (d) an amino acid sequence being encoded by a nucleic acid sequence being at least 80 % identical to the nucleotide sequence of (b), and (ii) a guide RNA; and (B) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA for degrading nucleic acid molecules in a sample; wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity.

The present invention relates in a eighth aspect to the use of (A) a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3; (b) an amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6 or 7; (c) an amino acid sequence being at least 80% identical to the amino acid sequence of (a), or (d) an amino acid sequence being encoded by a nucleic acid sequence being at least 80 % identical to the nucleotide sequence of (b), and (B) a guide RNA; and (C) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA for degrading nucleic acid molecules in a sample; wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity. The present invention relates in a ninth aspect to the use of (A) a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3; (b) an amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6 or 7; (c) an amino acid sequence being at least 80% identical to the amino acid sequence of (a), or (d) an amino acid sequence being encoded by a nucleic acid sequence being at least 80 % identical to the nucleotide sequence of (b), and (B) a nucleic acid molecule encoding in expressible form a guide RNA; and (C) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA for degrading nucleic acid molecules in a sample; wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity.

The present invention relates in a tenth aspect to the use of (A) a nucleic acid molecule encoding in expressible form a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3; (b) an amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6 or 7; (c) an amino acid sequence being at least 80% identical to the amino acid sequence of (a), or (d) an amino acid sequence being encoded by a nucleic acid sequence being at least 80 % identical to the nucleotide sequence of (b), and (B) a guide RNA; and (C) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA for degrading nucleic acid molecules in a sample; wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity.

The above uses of the sixth to tenth aspect of the invention are related to the methods of the first to fifth aspect of the invention. The above definitions and preferred embodiments of the first to fifth aspect of the invention therefore apply mutatis mutandis to the sixth to tenth aspect, as far as being applicable with the uses of sixth to tenth aspect. Also in connection with the uses the three components of a CRISPR nuclease having nonspecific nuclease activity, a guide RNA and an activation RNA are employed together for degrading the nucleic acid molecules in a sample. As explained above, the guide RNA and the activation RNA activate the nonspecific nuclease activity CRISPR nuclease having nonspecific nuclease activity and this activated CRISPR nuclease then degrades the nucleic acid molecules in a sample.

Since the uses are not confined to a particular timely order of steps it is to be understood that the uses cover both options: (I) the CRISPR nuclease having nonspecific nuclease activity is activated within the sample and (II) the CRISPR nuclease having nonspecific nuclease activity is preactivated and then added in its active form to the sample.

The five uses differ from each other regarding the form of the gRNA and the CRISPR nuclease just as the five methods. In the sixth aspect the gRNA and the CRISPR nuclease are complexed with each other and thereby form a ribonucleoprotein complex (RNP). In the seventh aspect the one or more nucleic acid molecules are used that encode in expressible form the CRISPR nuclease having nonspecific nuclease activity and the guide RNA. In the eight aspect the gRNA is used as RNA and the CRISPR nuclease as protein (but they are not complexed together to from the RNP). In the ninth aspect the CRISPR nuclease is used as protein and a nucleic acid molecule encoding in expressible form a guide RNA. Finally, in the tenth aspect a nucleic acid molecule encoding in expressible form the CRISPR nuclease is used and the guide RNA as RNA.

In accordance with a preferred embodiment of all above aspects of the invention, wherein the method or use is an in vitro or ex vivo method or use.

An ex vivo method or use is a method or use being carried out of the context of a living organism. Similarly, in vitro methods or uses are performed with microorganisms, cells, or biological molecules outside their normal biological context.

Both, in vitro and ex vivo methods exclude methods for treatment of the human or animal body by surgery or therapy and diagnostic methods practised on the human or animal body.

In accordance with another preferred embodiment of all above aspects of the invention, the PAM sequence is TTN, wherein N is T, A, G or C.

The PAM sequence is not the same for all classes of CRISPR nucleases and the exact PAM sequence varies. Hence, the same recognized PAM sequences is not shared by all CRISPR nucleases. The BEC family specific PAM sequence is TTN, wherein N is T, A, G or C.

In accordance with a further preferred embodiment of all above aspects of the invention, the sample is an industrial composition, food or beverage composition, a cosmetic composition, a diagnostic composition or a pharmaceutical composition.

As discussed herein above, the presence of nucleic acid molecules and in particular biologically active nucleic acid molecules is often undesired. Non-limiting but preferred examples of biologically active nucleic acid molecules are genomic DNA, mRNA, tRNA, miRNA, non-coding RNA or viral RNA or DNA is unwanted.

In this connection “biologically-active” means that those nucleic acid molecules affect biological processes (beyond the nutritional value) in a way which has an impact on living biological matter, such as cells or an organism. While the effect on living matter can be beneficial or adverse, the effect is preferably adverse.

The industrial composition may be any composition that is used in the industrial sector of the economy, including agriculture. The industrial sector includes manufacturing, mining, and utilities.

The food composition is any composition that applies any nutritious substance to a human or animals. The food composition can be eaten or drunken.

The beverage composition is any composition that is used as a drink, for the purpose of relieving thirst and introducing fluids in the body, nourishing the body and stimulating or soothing an individual.

The cosmetic composition is a composition being intended to be applied onto the consumer's skin, particularly onto the facial skin, such as onto the facial skin area surrounding the eyes, so as to regulate the condition of the skin and/or to improve the appearance of the skin.

A diagnostic composition is intended to be used in the diagnosis or a disease or condition. For instance, cells wherein a gene encoding a particular fluorescent protein of interest has been introduced, this protein may be used in diagnosis since it can be detected within an organism or a tissue sample. The term “pharmaceutical composition” relates to a composition for administration to a patient, preferably a human patient. The pharmaceutical composition to be used in the invention comprises the cells recited above. It may, optionally, comprise further molecules capable of altering the characteristics of the cells of the invention thereby, for example, stabilizing, modulating and/or activating their function. The composition is preferably in liquid form, e.g. (a) solution(s). The pharmaceutical composition of the present invention may, optionally and additionally, comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, organic solvents etc. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The therapeutically effective amount for a given situation will readily be determined by routine experimentation and is within the skills and judgement of the ordinary clinician or physician.

In accordance with another preferred embodiment the sample is a naturally occurring sample and preferably a body sample, plant sample, water sample or soil sample.

The body sample can be a body fluid or tissue sample. Body fluid are samples that were isolated or derived from liquids within the human body. Non-limiting but preferred examples of body fluids are blood (e.g. whole blood, serum or plasma), salvia, mucus, urine, semen, vaginal fluid and cerebrospinal fluid. Tissue samples may be derived from organs, such as skin, liver, heart, colon, stomach or kidney.

The plant sample may be or may derived from a whole plant or plant parts. Non-limiting but preferred examples of plant parts are leaves, callus, seed, fruits, flowers, roots and stems.

The water sample or soil sample can be taken, for example, from any natural environment. Non-limiting but preferred examples of water sample are tap water, sea, lake or river water, brackish water and rainwater. The solid sample may be obtained, for example, from building land, an agricultural area or a public ground, such as sports field, playing ground or park. According to a further preferred embodiment of all the above aspects of the invention the sequence identity of at least 80% is at least 85%, preferably at least 90% and most preferably at least 95%.

Also described and covered herein are sequence identities of at least 96%, at least 97%, at least 98% and at least 99%. Amino acid sequence as well as nucleotide sequence analysis and alignments in connection with the present invention are preferably carried out using the NCBI BLAST algorithm (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). The skilled person is aware of additional suitable programs to align nucleic acid sequences.

Also in connection with this preferred embodiment it is to be understood that the CRISPR nucleases to be used herein are not only structurally defined but are in addition functionally defined as having the nonspecific nuclease activity as detailed herein above.

Finally, according to a preferred embodiment of all the above aspects of the invention the method and uses may further comprise the removal of the degraded nucleic acid molecules from the sample.

Suitable means and method for removing fragments of nucleic acid molecules from a sample are know in the art and include, for example, centrifugation and affinity column chromatography. Spin-columns combine centrifugation and affinity column chromatography.

Regarding the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise. Similarly, and also in those cases where independent and/or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1 , a dependent claim 2 referring back to claim 1 , and a dependent claim 3 referring back to both claims 2 and 1 , it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1 , of claims 4, 2 and 1 , of claims 4, 3 and 1 , as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

The figures show.

Figure 1 - Gel electrophoresis visualizing the collateral DNA and RNA degradation of the BEC10 nuclease after the initial activation using a specific activation RNA.

Figure 2 - Gel electrophoresis visualizing the speed of the collateral dsDNA degradation of a plasmid using the BEC10 nuclease after the initial activation using a specific activation RNA.

The examples illustrate the invention.

Example 1 : Construction of a two-vector system for the expression of the BEC10 nuclease (CRISPR/BEC-FLAG-Ec) and the gRNA (CRISPR/gRNA-Ec) in E. coli

1.1 CRISPR/BEC-FLAG-Ec and CRISPR/gRNA-Ec vector systems for BEC expression (E. coli) and RNP purification

The necessary genetic elements for inducible expression and FLAG tag purification of the BEC10 nuclease and for the constitutive expression of the guide RNA (gRNA) transcription were provided on two separate vectors (CRISPR/BEC10-FLAG-Ec and CRISPR/gRNA-Ec).

In the following, the construction of the CRISPR/BEC10-FLAG-Ec vector and the CRISPR/gRNA-Ec systems are described.

Design of the CRISPR/BEC-FLAG-Ec protein expression vector

The synthetic 3696 bps BEC10 nucleotide sequence was codon optimized for expression in E. coli BW25113, using a bioinformatics application provided by the gene synthesis provider GeneArt (Thermo Fisher Scientific, Regensburg, Germany). For protein expression, the resulting synthetic gene was fused to the inducible araC-ParaBAD inducible promoter system and the fdT terminator (Otsuka & Kunisawa, Journal of Theoretical Biology 97 (1982), 415- 436).

Additionally, the DNA nuclease coding sequence was 3’ extended by a sequence encoding a nucleoplasmin nuclear localization signal (NLS) and two SV40 NLS. At the 5’ end of the DNA nuclease coding sequence, the sequence was extended by a sequence encoding a myc NLS. For purification of the DNA nuclease a FLAG tag (DYKDDDDK) was linked by a linker to the myc NLS to the 5’ coding sequence of the DNA nuclease.

The final BEC10_E. coli protein expression cassette was inserted by Gibson Assembly Cloning (NEB, Frankfurt, Germany) into an E. coli shuttle vector, containing all necessary genetic elements for episomal propagation and selection of recombinant E. coli cells.

CRISPR/BEC10-FLAG-Ec vector system

The complete nucleotide sequence of the constructed CRISPR/BEC10-FLAG-Ec vector system is provided as SEQ ID NO: 8.

Design of the guide RNA (gRNA) expression vector

The expression of the chimeric gRNA for target specific activation of the BEC10 nuclease was driven by the SacB RNA polymerase II promoter from Bacillus megaterium (Richhardt et al., Applied Microbiology Biotechnology 86 (2010), 1959-1965) and terminated using the transcription T1 and T2 termination region of the E. coli rrnB gene (Orosz et al., European Journal of Biochemistry 201 (1991), 653-659). The chimeric gRNA was composed of a constant 19 bps BEC family Stem-Loop sequence (SEQ ID NO: 9) fused to a target-specific 21 bps spacer sequence (SEQ ID NO: 10) matching a specific target sequence located on the activation-RNA.

The final gRNA expression cassette was inserted by Gibson Assembly Cloning (NEB, Frankfurt, Germany) into an E. coli shuttle vector, containing all necessary genetic elements for episomal propagation and selection of recombinant E. coli cells.

The construction of the final CRISPR/gRNA-Ec vector system was mediated by Gibson Assembly Cloning (NEB, Frankfurt, Germany).

The identity of all cloned DNA elements was confirmed by Sanger-Sequencing at LGC Genomics (Berlin, Germany). The complete nucleotide sequence of the constructed CRISPR/gRNA-Ec vector system is provided as SEQ ID NO: 11 .

1 .2 E. coli cultivation and transformation

Transformation of competent E. coli BW25113 cells

In brief, a single colony of E. coli BW25113 was inoculated in 5 ml LB medium and incubated for 12 to 14 h at 37 °C on a horizontal shaker at 200 rpm. Overnight grown pre-cultures were diluted into fresh 60 ml LB medium to an optical density at 600 nm (OD600) of 0.06. The inoculated medium was incubated at 30 °C on a horizontal shaker at 200 rpm until the culture reached an optical density at OD600 of 0.2. 600 pl 20 % arabinose was added and the cells were incubated at 30 °C at 200 rpm until the culture reached an optical density at OD600 of 0.5. Cells were transferred into a 50 ml conical tube and harvested by centrifugation at 4 °C for 5 min and 4000 x g. Pelleted cells from 50 ml culture were resuspended in 60 ml water and centrifuged at 4 °C for 5 min and 4000 x g.

A washing procedure was performed and the cells were resuspended in 30 ml 10 % glycerin following a centrifugation at 4 °C for 5 min and 4000 x g. In a second washing step, the cells were resuspended in 6 ml 10 % glycerin following a centrifugation at 4 °C for 5 min and 4000 x g. In the final step, the cells were resuspended in 150 pl 10 % glycerin. Aliquots of 25 pl competent cells were stored at - 80°C until use. For transformation procedure, aliquots of competent cells were thawed and 100 ng plasmid DNA (CRISPR/gRNA vectors) was added. Prepared cells were electroporated using 1800 V, 25 pF, 200 Ohm for 5 msec. Subsequently, 975 pL of NEB® 10-beta/Stable Outgrowth Medium was added and 25 pl of the suspension was plated on selective agar plates.

Nuclease expression

In brief, a single colony of E. coli BW25113 + CRISPR/BEC10-FLAG-Ec + CRISPR/gRNA-Ec was inoculated in 5 ml LB-Kan-Amp medium and incubated for 12 to 14 h at 37 °C on a horizontal shaker at 200 rpm. The overnight grown pre-cultures were diluted into fresh 60 ml LB medium to an optical density at 600 nm (OD600) of 0.05. The inoculated medium was incubated at 37 °C on a horizontal shaker at 200 rpm until the culture reached an optical density at OD600 of 0.2. The incubation temperature was lowered to 21 °C. At an optical density OD600 of 0.5 600 pl 20 % L-arabinose was added and the cells were incubated at 21 °C at 200 rpm for approximately 21 hours until the culture reached an optical density at OD600 of 4. Cells were transferred into a 50 ml conical tube and harvested by centrifugation at 4 °C for 10 min and 4000 x g. Pelleted cells from 90 ODV culture were stored at - 20 °C.

Cell disruption and FLAG tag purification

90 ODV pelleted cells were resuspended in 3 ml lysis buffer (50mM NaPi pH7, 100 mM NaCI + 1 tablet of complete™ EDTA free protease inhibitor cocktail per 50 ml) and disrupted by ultrasonic cell disruption using Branson Sonifier 250 with four ultrasonic cycles with 50 % duty cycle, 2.5 output for 30 sec. Between each cycle the cells were cooled down for 1 min on ice. The soluble fraction was separated from the insoluble fraction by centrifugation at 4 °C for 10 min and 4500 rpm.

The nuclease and gRNA (already coupled to each other) were purified by Anti-FLAG® M2 magnetic beads (Sigma Aldrich, St. Louis, USA). 20 pl magnetic bead aliquots were used for purification of 500 pl soluble cell fraction. The magnetic beads were equilibrated two times with 200 pl TBS buffer (50 mM T ris-HCI pH7.5, 150 mM NaCI). The supernatant was removed using a magnetic stand. 500 pl soluble fraction was incubated with the magnetic beads for 18 - 20 hours on a rotator at 4 °C. After binding, the magnetic beads were washed three times with 500 pl TBS buffer and the supernatant was removed in a magnetic stand. The nuclease/gRNA complex was eluted from the beads three times by the addition of each 50 pl FLAG protein (150 ng/pl) solved in TBS buffer and incubated for 30 min at room temperature on a rotator. The supernatant containing the purified ribonucleoprotein (RNP) was removed from the beads in the magnetic stand.

In vitro transcription of the activation-RNA

To pre-activate the BEC nuclease a synthetic activation-RNA was created encompassing the BEC family specific PAM sequence (TTN) and a 21 bp protospacer sequence. The activation- RNA was transcribed in vitro with HiScribe™ T7 Quick High Yield RNA Synthesis Kit and purified using Monarch® RNA Clean Up Kit (50 pg) (NEB, Frankfurt, Germany) according to the user protocol.

The complete nucleotide sequence of the constructed activation-RNA is provided as SEQ ID NO: 12.

In vitro activity assay

The collateral activity of the BEC10 RNPs (activated by an activation-RNA) was tested in vitro. As the degradation template a filter-sterilized supernatant of a B. subtilis fermentation was used for the in vitro reaction. 20 pl in vitro reaction encompassing 25 pmol RNPs (nuclease/gRNA complex), 25 pmol activation-RNA and 1 pl B. subtilis fermentation supernatant in 1x NEB 3.1 buffer were incubated at 37 °C for 1 h. The degradation of genomic DNA in the Bacillus supernatant by BEC10 was visualized with a 1 % agarose gel.

Example 2: Usage of the collateral activity of BEC family nucleases to deplete DNA and RNA from fermentation broth or supernatant

As described in WO2022017633 A2, BEC family nucleases are a new type of class 2 nucleases with a novel mechanism (collateral activity) to target DNA & RNA, which can be used for genome editing in combination with a homology directed repair template. Apart from genome editing applications, this novel mode of action can also be used in various application areas where classical CRISPR nucleases are not suitable. Here we demonstrate the usage of BEC family nucleases for the efficient depletion of DNA and RNA from fermentation broth or supernatant.

Experimental Setup:

The in vitro activity assay was carried out like described in Example 1 and the agarose gel to visualize the results is shown in Fig. 1.

25 pmol BEC10 RNPs, 25 pmol of activation-RNA (contains a BEC specific PAM sequence and a protospacer sequence matching the spacer sequence of the gRNA) and 1 pl B. subtilis fermentation supernatant were mixed in 1x NEB 3.1 buffer and incubated for 1 h at 37 °C.

As a negative control, the same experimental setup was used replacing the activation-RNA with a non-activating-RNA which does not contain a protospacer sequence matching the spacer sequence of the gRNA.

Results:

As illustrated in the negative control (Figure 1 ; Lane *) the fermentation supernatant which was incubated with a non-activated BEC10 nuclease contains a broad range of nucleotide sequences ranging from -500 bp - 20.000 bps . Furthermore, HMW DNA is detectable in the loading pocket of the agarose gel.

In contrast to this, the HMW DNA as well as the nucleotide sequences ranging from -500 bp - 20.000 bps are significantly degraded when incubated together with the pre-activated BEC10 nuclease (Figure 1 ; Lane **), demonstrating the ability of BEC family nuclease to deplete DNA and RNA from fermentation broth and supernatant.

Conclusion:

The generated results demonstrate the ability of BEC family nucleases to nucleic acid molecules in a sample after an initial activation using a specific activation-RNA. This application is based on the novel mechanism of BEC family nucleases as revealed herein, which once activated by a specific PAM and protospacer sequence (guide RNA) present on the activation-RNA elicit collateral activity leading to the degradation of all nucleic acid molecules (in particular DNA and RNA) in a sample. Because of this novel mechanism, classical CRISPR nucleases are not suitable to work for DNA and RNA depletion and it is the first time that a RNA guided CRISPR nuclease is successfully used for this purpose.

Example 3: Time course of dsDNA degradation induced by the collateral activity of the BEC family nucleases

In vitro activity assay:

The collateral activity of the BEC10 RNPs (activated by an activation-RNA) was tested in vitro. As the degradation template a plasmid (SEQ ID NO: 13) that does not contain the target sequences for the BEC10 activation was used. 20 pl in vitro reaction composition encompassing 10 pmol RNPs (nuclease/gRNA complex), 1 pmol activation-RNA and 500 ng of the plasmid in 1x NEB 3.1 buffer were incubated at 37 °C for different time periods before the activity of the BEC10 nuclease was stopped by adding 4 pl Purple Loading Dye 6x (NEB B7024S) containing SDS und EDTA leading to the denaturation of the BEC10 nuclease. In addition, the samples where heat inactivated for 20 min at 65 °C. The degradation of the Plasmid dsDNA by BEC10 was visualized in a 1 % agarose gel shown in Fig. 2.

Results:

As illustrated in Fig. 2. in the negative control (right lane, no activation RNA added) the plasmid is visible as one distinct band at around 2.5 kbp which is shorter than the sequence length of the plasmid. This can be explained by the fact that the circular plasmid in its native form is supercoiled and superhelical twisting leads to a more compact structure of DNA; the greater the superhelical twisting (or supercoiling), the more compact the structure. Therefore, the more supercoiled the DNA molecule, the faster it will migrate through an agarose gel towards the cathode (Gibson et al., Methods Mol Biol., 2020). In contrast to this, the incubation together with the spacer specific activation RNA to activate the collateral activity of the BEC10 nuclease shows the rapid degradation of the plasmid. In detail, after one minute of incubation time the plasmid is still present as one distinct band but at around 4.4 kbp. This perfectly matches the sequence length of the plasmid (4.463 bp) most likely due to the linearization of the plasmid by the BEC10 nuclease. After four minutes the distinct plasmid band is already very weak and most of the plasmid DNA is shredded down to smaller pieces in a range between 0.5 - 2 kbp. Within 15 minutes of incubation time no visible plasmid band is detectable anymore and most of the DNA pieces are smaller than 200 bp demonstrating the rapid collateral dsDNA activity of the BEC family nucleases.

Conclusion:

The generated results demonstrate the rapid dsDNA collateral activity of BEC family nucleases leading to the unspecific degradation of dsDNA. In comparison to other nucleases with a collateral activity where a collateral activity on RNA (Cas13) or ssDNA (Cas12a) is described, BEC family nucleases are able to degrade RNA, ssDNA and dsDNA making them a group of more versatile and robust nucleases in many applications. As shown in example 2, BEC family nucleases are able to degrade all RNA & DNA species from a B. subtilis fermentation supernatant. As dsDNA is the most stable form of all nucleic acids it is the hardest to deplete from a fermentation broth and no other CRISPR nuclease would be applicable in this scenario, as they do not have a collateral activity targeting dsDNA. Therefore, BEC family nucleases have a clear advantage in comparison to other CRISPR nucleases with their unique ability to degrade dsDNA.

Claims

1 . A method for degrading nucleic acid molecules in a sample, comprising

(I) contacting the sample with (A) a ribonucleoprotein complex (RNP) comprising or consisting of

(i) a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of

(a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3;

(b) an amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6 or 7;

(c) an amino acid sequence being at least 80% identical to the amino acid sequence of (a), or

(d) an amino acid sequence being encoded by a nucleic acid sequence being at least 80 % identical to the nucleotide sequence of (b),

(ii) in complex with a guide RNA; and

(B) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity; wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity; or

(II)

(a) activating the nonspecific nuclease activity of a CRISPR nuclease having nonspecific nuclease activity by contacting the RNP as defined in (I) with the activation RNA or the nucleic acid molecule encoding in expressible form the activation RNA as defined in (I) under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity, and

(b) contacting the sample with the activated CRISPR nuclease having nonspecific nuclease activity.

2. A method for degrading nucleic acid molecules in a sample, comprising

(I) contacting the sample with

(A) one or more nucleic acid molecules, said one or more nucleic acid molecules encoding in expressible form

(a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3;

(d) an amino acid sequence being encoded by a nucleic acid sequence being at least 80 % identical to the nucleotide sequence of (b), and

(ii) a guide RNA; and

(II)

(a) activating the nonspecific nuclease activity of a CRISPR nuclease having nonspecific nuclease activity by contacting the one or more nucleic acid molecules as defined in (I) with the activation RNA or the nucleic acid molecule encoding in expressible form the activation RNA as defined in (I) under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity, and

3. A method for degrading nucleic acid molecules in a sample, comprising

(I) contacting the sample with

(A) a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of

(a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3;

(B) a guide RNA; and

(C) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity; wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity; or

(II)

(a) activating the nonspecific nuclease activity of a CRISPR nuclease having nonspecific nuclease activity by contacting the CRISPR nuclease having as defined in (I) with the guide RNA and the activation RNA or the nucleic acid molecule encoding in expressible form the activation RNA as defined in (I) under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity, and

4. A method for degrading nucleic acid molecules in a sample, comprising

(I) contacting the sample with

(A) a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3;

(B) a nucleic acid molecule encoding in expressible form a guide RNA; and

(II)

(a) activating the nonspecific nuclease activity of a CRISPR nuclease having nonspecific nuclease activity by contacting the CRISPR nuclease having as defined in (I) with the nucleic acid molecule encoding in expressible form the guide RNA and the activation RNA or the nucleic acid molecule encoding in expressible form the activation RNA as defined in (I) under conditions wherein the activation- RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity, and

5. A method for degrading nucleic acid molecules in a sample, comprising

(I) contacting the sample with

(A) a nucleic acid molecule encoding in expressible form a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of

(a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3;

(B) a guide RNA; and

(II)

(a) activating the nonspecific nuclease activity of a CRISPR nuclease having nonspecific nuclease activity by contacting the nucleic acid molecule encoding in expressible form the CRISPR nuclease having nonspecific nuclease activity as defined in (I) with the guide RNA and the activation RNA or the nucleic acid molecule encoding in expressible form the activation RNA as defined in (I) under conditions wherein the activation-RNA together with the guide RNA activates the CRISPR nuclease having nonspecific nuclease activity, and

6. Use of

(A) a ribonucleoprotein complex (RNP) comprising or consisting of

(i) a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3;

(ii) in complex with a guide RNA; and (B) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA for degrading the nucleic acid molecules in a sample; wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity.

7. Use of

(a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3;

(ii) a guide RNA; and

(B) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA for degrading nucleic acid molecules in a sample; wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity.

8. Use of

(A) a CRISPR nuclease having nonspecific nuclease activity comprising or consisting of (a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3; (b) an amino acid sequence being encoded by the nucleotide sequence of SEQ ID NO: 4, 5, 6 or 7;

(B) a guide RNA; and

(C) an activation RNA or a nucleic acid molecule encoding in expressible form the activation RNA for degrading nucleic acid molecules in a sample; wherein the guide RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the activation RNA and (b) a second segment that interacts with the CRISPR nuclease having nonspecific nuclease activity; and wherein the activation RNA comprises (a) a first segment comprising a nucleotide sequence that is complementary to a sequence of the guide RNA and (b) a second segment that is the protospacer adjacent motif (PAM) sequence of the CRISPR nuclease having nonspecific nuclease activity.

9. Use of

(a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3;

(B) a nucleic acid molecule encoding in expressible form a guide RNA; and

10. Use of

(a) an amino acid sequence of any one of SEQ ID NO: 1 , 2 or 3;

(B) a guide RNA; and

11 . The method of any one of claims 1 to 5 or the use of any one of claims 6 to 10, wherein the method or use is an in vitro or ex vivo method or use.

12. The method or use of any preceding claim, wherein the PAM sequence is TTN, wherein N is T, A, G or C.

13. The method or use of any preceding claim, wherein the sample is an industrial composition, food or beverage composition, a cosmetic composition, a diagnostic composition or a pharmaceutical composition.

14. The method or use of any preceding claim, wherein the sample is a naturally occurring sample and preferably a body sample, plant sample, water sample or soil sample.

15. The method or use of any preceding claim, wherein the sequence identity of at least 80% is at least 85%, preferably at least 90% and most preferably at least 95%.