PaCas9 nuclease

Abstract

The present invention relates to the fields of biotechnology, molecular biology and medicine, particularly to nuclease enzymes and their uses. More specifically, the present invention relates to PaCas9 nuclease enzyme. The present invention also relates to nucleic acids encoding the nuclease, genetic constructs comprising the nucleic acids, expression vectors, delivery vectors, liposomes comprising the nuclease or the nucleic acids encoding the nuclease, methods for producing the nuclease, methods for delivery, and host cells comprising the nucleic acids encoding the nuclease. [Selection diagram] None

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology, molecular biology and medicine, particularly to nuclease enzymes and their use. More specifically, the present invention relates to the PaCas9 nuclease enzyme. The present invention also relates to nucleic acids encoding said nuclease, gene constructs comprising said nucleic acids, expression vectors, delivery vectors, liposomes comprising said nuclease or nucleic acids encoding said nuclease, methods for producing the nuclease, methods for delivery, and host cells comprising nucleic acids encoding said nuclease.

Background Art

[0002] It was first shown in 2007 that CRISPR-Cas is an adaptive immune system in most of many bacteria and archaea (Barrangou et al., 2007, Science 315: 1709 - 1712; Brouns et al., 2008, Science 321: 960 - 964). Based on functional and structural criteria, three types of CRISPR-Cas systems have been characterized so far, most of which use small RNA molecules as guides targeting complementary DNA sequences (Makarova et al., 2011, Nat Rev Microbiol 9: 467 - 477; Van der Oost et al., 2014, Nat Rev Microbiol 12: 479 - 492).

[0003] In a recent study by the Daudna / Charpentier laboratory, complete characterization of the effector enzyme of the type II CRISPR-Cas system (Cas9) was performed, which included demonstration that introduction of a designed CRISPR RNA guide (containing a specific spacer sequence) targets a complementary sequence (protospacer) on a plasmid, causing a double-strand break in this plasmid (Jinek et al., 2012, Science 337: 816 - 821). Subsequently, Jinek et al., 2012 used Cas9 as a tool for genome editing.

[0004] Cas9 has been used to manipulate the genomes of a range of eukaryotic cells (e.g., fish, plants, humans) (Charpentier and Doudna, 2013, Nature 495: 50-51).

[0005] Furthermore, Cas9 has been used to improve the yield of homologous recombination in bacteria by selecting for specialized recombination events (Jiang et al., 2013, Nature Biotechnol 31: 233-239). To achieve this, a toxic fragment (targeting construct) is co-transfected with a rescue fragment (editing construct carrying point mutations or deletions) carrying the desired modification. The targeting construct consists of Cas9 combined with a designed CRISPR and an antibiotic resistance marker, which defines the desired recombination site on the host chromosome; in the presence of the corresponding antibiotic, integration of the targeting construct in the host chromosome is selected for. The host can escape autoimmunity problems only if further recombination of the editing construct containing the CRISPR target site occurs elsewhere on the host chromosome. Thus, in the presence of the antibiotic, only the desired (marker-free) mutants can survive and proliferate. Related strategies are also presented for selection regarding subsequent removal of the integrated targeting construct from the chromosome, which generates true marker-free mutants.

[0006] In recent years, CRISPR-Cas-mediated genome editing has been established as a useful tool for genetic manipulation. The prokaryotic CRISPR system functions as an adaptive immune system for its host (Jinek et al., 2012, Science 337: 816-821), and it has been established that this system can be used for rapid and efficient genetic manipulation (e.g., Mali et al., 2013, Nat Methods 10: 957-963), and that only modification of the guide sequence is required to target a sequence of interest.

Prior Art Documents

Non-Patent Literature

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Summary of the Invention

Problems to be Solved by the Invention

[0008] However, for applications in the fields of genetic research and genome editing, there is a continuing need for the development of agents involving improved sequence-specific nucleic acid detection, cleavage, and manipulation under diverse experimental conditions.

Means for Solving the Problems

[0009] The present invention relates to a PaCas9 nuclease having the amino acid sequence of SEQ ID NO: 2. In one aspect, the present invention relates to an isolated nucleic acid molecule encoding a PaCas9 nuclease having the nucleotide sequence of SEQ ID NO: 1.

[0010] In one aspect, the present invention relates to an expression vector comprising a nucleic acid having the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the expression vector is a gene construct as shown in FIG. 1, PpCas9-T2A-GFP-sgRNA1-MCS-sgRNA2-MCS.

[0011] In one aspect, the present invention relates to a vector for delivering a therapeutic agent comprising a nucleic acid having the nucleotide sequence of SEQ ID NO: 1. In one embodiment of the present invention, the vector delivers the therapeutic agent to a target cell or target tissue.

[0012] In one aspect, the present invention relates to a liposome for delivering a therapeutic agent comprising a PaCas9 nuclease having the amino acid sequence of SEQ ID NO: 2 and having the nucleotide sequence of SEQ ID NO: 1.

[0013] In one embodiment of the present invention, the liposome delivers the therapeutic agent to a target cell or target tissue. In one aspect, the present invention relates to a method for delivering a therapeutic agent to a target cell or target tissue using the above vector or the above liposome.

[0014] In one embodiment of the method, the therapeutic agent is delivered to a target cell or target tissue by administering the above vector or the above liposome into a mammalian body. In one aspect, the present invention relates to a method for producing a host cell producing a PaCas9 nuclease having the amino acid sequence of SEQ ID NO: 2, comprising transforming a cell using any of the above vectors.

[0015] In one aspect, the present invention relates to a method for producing PaCas9 nuclease, the method comprising culturing the host cell in a growth medium under conditions sufficient to produce the PaCas9 nuclease, and, if necessary, subsequently isolating and purifying the obtained PaCas9 nuclease. BRIEF DESCRIPTION OF THE DRAWINGS

[0016]

Figure 1

[0017] AmpR is the beta-lactamase gene that provides resistance to ampicillin, The CMV promoter is the promoter of the cytomegalovirus early gene, The Kozak sequence is intended to enhance the translation efficiency of the protein, The start codon is the start codon, NLS refers to the nuclear localization signal (NLS), PaCas9 is the nucleotide sequence of SEQ ID NO: 1 encoding the PaCas9 nuclease having the amino acid sequence of SEQ ID NO: 2, FLAG is the FLAG epitope sequence used for protein detection, GFP is the modified green fluorescent protein, TK pA refers to the thymidine kinase polyA signal sequence used to increase mRNA stability, F1 ori is the origin of replication that enables packaging of the phagemid into phage particles when co-transformed with a helper phage, polIII term+U6 promoter refers to a cassette for the expression of small RNA molecules, each cassette containing a U6 promoter and an RNA polymerase III terminator.

[0018] The pUC origin is the pUC origin of replication in bacteria.

Figure 2

Modes for Carrying Out the Invention

[0019] Definitions and General Methods Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0020] Furthermore, unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include singulars. Typically, the classifications and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medical and pharmaceutical chemistry, as well as the hybridization and chemistry of proteins and nucleic acids described herein are well known to those of ordinary skill in the art and are widely used. Enzyme reactions and purification methods are performed according to the manufacturer's instructions as commonly practiced in the art or as described herein.

[0021] “Mammal” refers to any animal classified as a mammal, including primates, humans, rodents, dogs, cats, cows, small cattle, horses, pigs, etc. Nuclease Nucleases are a broad group of enzymes that hydrolyze the phosphodiester bonds between nucleic acid subunits.

[0022] Depending on their specificity and activity, nucleases may be of the following types: exonucleases and endonucleases, ribonucleases and deoxyribonucleases, restrictases and several others. Restrictases are important elements in applied molecular biology.

[0023] PaCas9 nuclease relates to the type of deoxyribonuclease. When bound to at least one RNA molecule that recognizes the target sequence, PaCas9 nuclease can cleave DNA containing the target nucleic acid sequence.

[0024] PaCas9 nuclease contains two endonuclease domains, each of which creates a single-strand break, and when both are activated, creates a double-strand break.

[0025] PaCas9 nuclease is an effector enzyme of the type II CRISPR-Cas system (type 2 nuclease). PaCas9 nuclease can create a double-strand DNA break at a very specific recognition site (16 - 20 characters).

[0026] The DNA of PaCas9 nuclease is presented in SEQ ID NO: 1. The amino acid sequence of PaCas9 nuclease is presented in SEQ ID NO: 2. Figure 2 shows the amino acid sequence of PaCas9 nuclease including domain distribution.

[0027] PaCas9 nuclease associates with clustered regularly interspaced short palindromic repeats (CRISPR), as well as other adjacent components of the CRISPR-Cas system: crRNA and tracrRNA sequences.

[0028] The nucleotide sequence encoding tracrRNA is presented in SEQ ID NO: 3. The nucleotide sequence encoding the direct repeat DR is presented in SEQ ID NO: 4. crRNA consists of a target-dependent variable part and the direct repeat DR presented in SEQ ID NO: 4.

[0029] As used herein, the term "therapeutic agent" refers to a PaCas9 nuclease having the amino acid sequence of SEQ ID NO:2, or an isolated nucleic acid molecule that encodes the PaCas9 nuclease and has the nucleotide sequence of SEQ ID NO:1.

[0030] tracrRNA (trans-activating crRNA) is a small molecule trans-coding RNA. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a special bacterial and archaeal locus consisting of direct repeats with unique sequences (spacers) interspersed.

[0031] Nucleic acid molecule The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence", and "nucleotide sequence" are used interchangeably herein and mean the exact sequence of nucleotides, modified or unmodified, determining a nucleic acid fragment or region that contains or does not contain unnatural nucleotides, and being either double-stranded DNA or RNA, single-stranded DNA or RNA, or the transcription product of said DNA.

[0032] It should also be included herein that the present invention is not related to nucleotide sequences in their natural chromosomal environment, i.e., in their natural state. The sequences of the present invention are isolated and / or purified, i.e., they have been sampled directly or indirectly, for example by copying, and their environment has been modified at least in part. Thus, isolated nucleic acids obtained by recombinant genetics, for example of host cells, or obtained by chemical synthesis must also be mentioned herein.

[0033] An "isolated" nucleic acid molecule is one that has been identified and separated from at least one nucleic acid molecule impurity with which it is associated in the natural source of the nuclease nucleic acid. Isolated nucleic acid molecules are different from the forms or sets found in natural conditions. Thus, isolated nucleic acid molecules are different from the nucleic acid molecules that exist in cells under natural conditions. However, isolated nucleic acid molecules include, for example, nucleic acid molecules located in cells where an antibody is normally expressed if the nucleic acid molecule has a chromosomal location different from its location in cells under natural conditions.

[0034] The term "nucleotide sequence" includes its complement unless otherwise specified. Thus, a nucleic acid having a particular sequence must be understood to include its complementary strand containing the complementary sequence.

[0035] The term "regulatory sequence" refers to a DNA sequence necessary for the expression of a coding sequence that is ligated so as to be functional in a particular host organism. Regulatory sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0036] Nucleic acids are "operably linked" when they are arranged so as to be in a functional relationship with another nucleic acid sequence. For example, DNA of a presequence or a secretory leader sequence is operably linked to DNA of a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned to promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in the reading frame. However, enhancers do not have to be contiguous.

[0037] Vector As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded piece of DNA, into which an additional DNA segment may be ligated. In some embodiments, the vector is a viral vector, where an additional DNA segment may be ligated into the viral genome. In some embodiments, the vector is capable of autonomous replication in the host cell into which it has been introduced (e.g., a bacterial vector having a bacterial origin of replication site and an episomal mammalian vector). In a further embodiment, the vector (e.g., a non-episomal mammalian vector) may be integrated into the host cell genome upon introduction into the host cell and thereby replicated along with the host gene. Further, certain vectors are capable of directing the expression of a gene to which they are ligated such that the gene is functional. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0038] In one aspect, the invention relates to a vector suitable for the expression of any nucleotide sequence described herein. The invention relates to a vector comprising a nucleic acid molecule encoding a PaCas9 nuclease.

[0039] In some embodiments, the PaCas9 nuclease of the present invention is expressed by inserting DNA into an expression vector such that the gene is operably linked to the necessary expression control sequences, such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The DNA molecule may be ligated into the vector such that the transcriptional and translational control sequences within the vector perform their intended function of controlling the transcription and translation of the DNA. The expression vector and the expression control sequences may be selected to be compatible with expression in the host cell used. The DNA molecule may be introduced into the expression vector by standard methods (e.g., ligation of the PaCas9 nuclease gene fragment and complementary restriction sites on the vector, or blunt-end ligation if no restriction sites are present).

[0040] In addition to the PaCas9 nuclease gene, the recombinant vector expression of the present invention may possess a control sequence that regulates the expression of the PaCas9 nuclease gene in a host cell. It will be understood by those skilled in the art that the design of the expression vector, including the selection of the control sequence, may depend on factors such as the choice of host cell to be transformed and the expression level of the desired protein. Preferred regulatory sequences for expression host cells in mammals include viral elements that ensure high-level protein expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., major late promoter of adenovirus (AdMLP)), promoters and / or enhancers derived from polyomavirus, as well as strong mammalian promoters, such as natural immunoglobulin promoters or actin promoters. For further description of viral regulatory elements and their sequences, see, for example, U.S. Patent Nos. 5,168,062, 4,510,245, and 4,968,615. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

[0041] In addition to the PaCas9 nuclease gene and control sequences, the recombinant expression vectors of the present invention may also possess additional sequences such as sequences that control the replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically, the selectable marker gene confers resistance to a medicinal agent, such as G418, hygromycin, or methotrexate, on the host cells into which the vector has been introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr− host cells during methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

[0042] The term "expression control sequence" is intended herein to refer to polynucleotide sequences necessary to achieve expression and processing of the coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., the Kozak consensus sequence); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences differs depending on the host organism; in prokaryotes, such control sequences generally include the promoter of the ribosome binding site and transcription termination sequences; in eukaryotes, such control sequences typically include a promoter and transcription termination sequences. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fusion partner sequences.

[0043] Host cell The term "recombinant host cell" (or simply "host cell") is intended herein to refer to a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells, which may contain, for example, the vectors described above for the present invention. It should be understood that "recombinant host cells" and "host cells" are intended to refer not only to particular target cells, but also to the progeny of such cells. Modifications can occur in subsequent generations due to either mutations or environmental influences, so such progeny may not actually be identical to the parental cell, but such cells are still included within the scope of the term "host cell" herein.

[0044] The nucleic acid molecules encoding the PaCas9 nuclease of the present invention and vectors containing these nucleic acid molecules may be used for transfection of suitable mammals or their cells, plants or their cells, bacteria or yeast host cells. Transformation may be performed by any known technique for the introduction of polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, cationic polymer-nucleic acid complex transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, encapsulation of polynucleotide(s) in liposomes, and direct microinjection of DNA into the nucleus. Additionally, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods for transfecting cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods for transforming plant cells are well known in the art and include, for example, Agrobacterium-mediated transformation, particle gun transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0045] Mammalian cell lines used as hosts for transformation are well known in the art and include a number of available immortalized cell lines. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, FreeStyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, and many other cell lines. The cell line is selected by determining which cell line has a high expression level and provides the necessary properties for the protein to be produced. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells. When introducing a recombinant expression vector encoding PaCas9 nuclease into a mammalian host cell, PaCas9 nuclease is produced by culturing the host cell for a sufficient period of time to allow expression of PaCas9 nuclease in the host cell or, more preferably, secretion of PaCas9 nuclease into the medium in which the host cell is grown. PaCas9 nuclease may be isolated from the medium using standard protein purification techniques. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, maize, wheat, potato, etc. Bacterial host cells include Escherichia coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0046] Furthermore, many known techniques may be used to enhance the production level of the PaCas9 nuclease of the present invention from the resulting cell line. For example, the glutamine synthetase gene expression system (GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in its entirety or in part in relation to EP Nos. 0216846, 0256055, 0323997, and 0338841.

[0047] PaCas9 nucleases obtained from different cell lines or transgenic animals are likely to have different glycosylation profiles when compared to each other. However, the PaCas9 nuclease encoded by the nucleic acid molecules described herein is part of the present invention regardless of its glycosylation state and generally regardless of the presence or absence of post-translational modifications.

[0048] Liposome In one aspect, the present invention relates to liposomes encapsulating a PaCas9 nuclease having the amino acid sequence of SEQ ID NO: 2, or to an isolated nucleic acid molecule encoding a PaCas9 nuclease and having the nucleotide sequence of SEQ ID NO: 1.

[0049] A liposome is a microscopic closed vesicle having an internal phase surrounded by one or more lipid bilayers and having the ability to retain water-soluble substances in the internal phase and lipid-soluble substances in the phospholipid bilayer. When encapsulating an active compound in a liposome and delivering it to a target tissue, methods for efficiently encapsulating the active compound in the liposome and ensuring stable retention of the active compound by the liposome constitute an important issue.

[0050] Generally, liposomes are considered particles having a main size ranging from several tens of nanometers to a fraction of a micron, and their shell accommodates molecules of another substance(s). The liposome shell is "semi-permeable" to water molecules and ions.

[0051] Liposomes are characterized by their ability to contain and retain substances of different properties. The range of substances incorporated into liposomes is very wide, spanning from inorganic ions and low-molecular-weight organic compounds to giant proteins and nucleic acids.

[0052] Liposomes provide for the long-term release of substances incorporated into the carrier. Liposomes may be made of phospholipids, particularly phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin, egg / soybean phospholipids or mixtures thereof. Non-limitingly, the present invention includes the following aspects. [Aspect 1] A PaCas9 nuclease having the amino acid sequence of SEQ ID NO: 2. [Aspect 2] An isolated nucleic acid molecule encoding the PaCas9 nuclease described in Aspect 1 and having the nucleotide sequence of SEQ ID NO: 1. [Aspect 3] An expression vector comprising the nucleic acid described in Aspect 2. [Aspect 4] The expression vector described in Aspect 3, which is the gene construct shown in FIG. 1. [Aspect 5] A vector for delivering a therapeutic agent comprising the nucleic acid described in Aspect 2. [Aspect 6] The vector described in Aspect 5, wherein the therapeutic agent is delivered to a target cell or target tissue. [Aspect 7] A method for delivering a therapeutic agent to a target cell or target tissue using the vector described in Aspects 3 to 6. [Aspect 8] A method for producing a host cell for producing the PaCas9 nuclease described in Aspect 1, the method comprising the step of transforming a cell with the vector described in any one of Aspects 3 to 6. [Aspect 9] A host cell for producing the PaCas9 nuclease described in Aspect 1, comprising the nucleic acid described in Aspect 2. [Aspect 10] A method for producing the PaCas9 nuclease described in Aspect 1, comprising culturing the host cell described in Aspect 9 in a growth medium under conditions sufficient to produce the PaCas9 nuclease, and then, if necessary, isolating and purifying the resulting PaCas9 nuclease.

Example

[0053] The following examples are provided to better understand the present invention. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention in any way.

[0054] All publications, patents, and patent applications cited herein are incorporated herein by reference. The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, but it will be apparent to those skilled in the art that certain changes and modifications may be made without departing from the spirit and scope of the appended claims in view of the description of the invention.

[0055] Materials and General Methods Recombinant DNA Technology Standard methods for manipulating DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 were used. Molecular biology reagents were used according to the manufacturer's instructions.

[0056] Gene Synthesis A desired gene segment was prepared from oligonucleotides produced by chemical synthesis. Gene segments 300 - 4000 kb in length flanked by specific restriction sites were assembled by annealing and ligation of oligonucleotides including PCR amplification, and subsequently cloned through the indicated restriction sites. The DNA sequence of the subcloned gene fragment was confirmed by DNA sequencing.

[0057] DNA sequencing The DNA sequence was determined by Sanger sequencing. DNA and protein sequence analysis and sequence data management For sequence generation, mapping, analysis, annotation and illustration, Infomax's Vector NTI Advance suite, version 8.0 was used.

[0058] Expression vector For the expression of PaCas9 nuclease, variants of expression plasmids intended for expression in prokaryotic cells (E. coli) and transient expression in eukaryotic cells (e.g., in CHO cells) were used. In addition to the PaCas9 nuclease expression cassette, the vector contained: an origin of replication enabling replication of the plasmid in E. coli, and a gene conferring resistance to E. coli against various antibiotics (e.g., against ampicillin and / or kanamycin).

[0059] Example 1 Method for preparing PaCas9 nuclease To prepare a metagenomic sequence, a Homoeodictya palmata sponge sample was collected from the White Sea region, the material was fractionated by centrifugation, then total DNA was isolated and subsequently sequenced.

[0060] Using bioinformatics methods, the open reading frame of the PaCas9 protein and adjacent components of the CRISPR-Cas system (CRISPR cassette, as well as crRNA and tracrRNA sequences) were detected in the metagenomic sequences.

[0061] The DNA of the PaCas9 nuclease is presented in SEQ ID NO: 1. The amino acid sequence of the PaCas9 nuclease is presented in SEQ ID NO: 2. The nucleotide sequence encoding tracrRNA is presented in SEQ ID NO: 3. The nucleotide sequence encoding the direct repeat DR is presented in SEQ ID NO: 4.

[0062] Example 2 Description of cloning By bioinformatics search, the PaCas9 nuclease gene sequence was obtained. The sequence was codon-optimized to ensure optimal expression in mammalian cells and then de novo assembled from chemically synthesized oligonucleotides using the Gibson method. The synthetic PaCas9 gene was cloned into the gene construct from the 3' end of the CMV promoter. A Kozak sequence and a nuclear localization signal (NLS) were added from the 5' end of the gene, and a FLAG epitope sequence for protein detection was added from the 3' end. The PaCas9 sequence and the related elements listed above, the T2A element, and the open reading frame of green fluorescent protein (EGFP) as an expression marker were placed in the construct in the same reading frame.

[0063] After the reading frame, a thymidine kinase polyA signal sequence is arranged from the 3'-end to increase the stability of the mRNA. In the region of the bacterial cortex of the gene construct for the expression of the small molecule RNA molecule, there are two tandem cassettes. Each cassette contains a U6 promoter and an RNA polymerase III transcription terminator. These cassettes are necessary for the expression of an RNA molecule that provides a specific interaction between the target DNA molecule (cellular genome) and the PaCas9 protein. The construct map is shown in Figure 1. This construct expresses both the PaCas9 protein (transported to the nucleus through the NLS) and the RNA molecule that guides the protein (guide RNA), enables the detection of the protein by the FLAG epitope, and determines the delivery efficiency of the gene construct by the detection of EGFP.

[0064] Example 3 Enzymatic activity of PaCas9 protein By comparing the homology of the HNH and RuvC domains of various Cas9 family proteins with the PaCas9 domain (the domain distribution is shown in Figure 2), the amino acids involved in the enzymatic hydrolysis of DNA / RNA were identified. The conserved amino acids previously shown to be involved in the enzymatic activity of the Cas9 protein were isolated in PaCas9. Therefore, by the analytical method, it was found that the amino acid residues of this protein are necessary for the enzymatic activity of the PaCas9 protein (amino acid positions): D9; E527; H750; D753; H613; N636.

[0065] Example 4 Determination of the enzymatic activity of PaCas9 protein To determine the PAM (protospacer adjacent motif) sequence, the inventors performed an in vitro reaction on a cut DNA library using a recombinant nuclease protein (SEQ ID NO: 2), a crRNA (consisting of a target-dependent variable part and a direct repeat presented in SEQ ID NO: 4), and a tracrRNA (SEQ ID NO: 3). The DNA library is a PCR fragment containing a 7-character randomized sequence and a recognizable sequence, the protospacer.

[0066] After incubating the PaCas9-RNA-protein complex with a DNA library, the reaction products are loaded onto a gel for electrophoresis. The uncut fragments are extracted from the gel and sequenced on an Illumina platform. Comparison of the PAM sequences contained in the uncut PaCas9 reaction products and the control reactions will enable determination of the PAM of the protein in question.

[0067] After identifying the PAM sequence, in vitro nuclease activity is evaluated. For this purpose, the protein complexed with the RNA guide is incubated with a DNA fragment possessing the protospacer sequence and the identified PAM. The optimal ratio of the RNA-protein complex to the cut DNA is determined. Nuclease activity is evaluated based on the amount of PaCas9 protein required for 50% cleavage of 200 ng of target DNA approximately 400 base pairs in length containing the optimal PAM.

[0068] Therefore, it was confirmed that PaCas9 nuclease has enzymatic activity and creates double-strand breaks in DNA. Furthermore, it was confirmed that PaCas9 nuclease can create double-strand breaks in DNA with a very specific recognition site (16 - 20 characters).

Claims

1. PaCas9 nuclease having the amino acid sequence of SEQ ID NO:

2.

2. An isolated nucleic acid molecule encoding the PaCas9 nuclease according to Claim 1 and having the nucleotide sequence of SEQ ID NO:

1.

3. An expression vector containing the nucleic acid according to Claim 2.

4. Beta-lactamase gene, Promoter of cytomegalovirus early gene, Kozak sequence, Start codon, Nucleic acid encoding a nuclear localization signal (NLS), Nucleotide sequence of SEQ ID NO: 1 encoding PaCas nuclease having SEQ ID NO: 2, Nucleic acid encoding a FLAG epitope sequence, Nucleic acid encoding a modified green fluorescent protein, Thymidine kinase poly A signal sequence, Replication origin, A cassette for the expression of guide RNA, each cassette comprising a U6 promoter and an RNA polymerase III terminator, and pUC replication origin in bacteria, The expression vector according to Claim 3, which is a gene construct comprising.

5. A vector for delivering a therapeutic agent containing the nucleic acid according to Claim 2, the vector containing the nucleic acid according to Claim 2.

6. The vector according to Claim 5, wherein the therapeutic agent is delivered to a target cell or a target tissue.

7. The vector according to any one of Claims 3 to 6, for use in the delivery of a therapeutic agent to a target cell or a target tissue.

8. A method for producing a host cell for producing a PaCas9 nuclease according to claim 1, the method comprising the step of transforming the cell with the vector according to any one of claims 3 to 6. **Claim 9** A host cell for producing a PaCas9 nuclease according to claim 1, comprising the nucleic acid according to claim 2. **Claim 10** A method for producing a PaCas9 nuclease according to claim 1, the method comprising culturing the host cell according to claim 9 in a growth medium under conditions sufficient to produce the PaCas9 nuclease, and, if necessary, subsequently isolating and purifying the obtained PaCas9 nuclease.

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