Cas enzyme and system and use thereof

Abstract

The present application relates to the field of biomedicine, and specifically relates to a novel Cas enzyme, a system thereof and the use thereof.

Description

Cas enzymes and their systems and applications Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a Cas enzyme and its system and application. Background Art

[0002] Recent advances in genome sequencing technologies and analytical methods have significantly accelerated our understanding of the genetic basis of biological activity across diverse domains, from prokaryotic biosynthetic pathways to human pathologies. To fully understand and evaluate the vast amounts of information generated by genome sequencing, corresponding improvements in the scale, efficiency, and ease of use of genome and epigenome manipulation technologies are needed. These new genome and epigenome engineering technologies will accelerate the development of new applications across many fields, including biotechnology, agriculture, and human therapeutics.

[0003] Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes, collectively referred to as CRISPR-Cas or CRISPR / Cas systems, are now understood to provide immunity to phage infection in bacteria and archaea. The CRISPR-Cas system of prokaryotic adaptive immunity is an extremely diverse set of protein effectors, noncoding elements, and locus architectures, some of which have been engineered and adapted to produce important biotechnological results. Components of the system involved in host defense include one or more effector proteins capable of modifying DNA or RNA and RNA guide elements responsible for targeting the activity of these proteins to specific sequences on phage DNA or RNA, which can be reprogrammed to target alternative DNA or RNA targets.

[0004] CRISPR-Cas systems can be roughly divided into two categories: Class 1 systems are composed of multiple effector proteins, and Class 2 systems are composed of a single effector protein, which is complexed with an RNA guide to target DNA or RNA substrates. The single-subunit effector composition of Class 2 systems provides a simpler set of components for engineering and application conversion, and has been an important source of programmable effectors to date. The characterization and engineering of Class 2 CRISPR-Cas systems, such as CRISPR-Cas9, have paved the way for diverse and extensive biotechnology applications in genome editing and other areas. However, in addition to the current CRISPR-Cas systems that have achieved new applications through their unique properties, there is still a need to develop powerful genome engineering tools, that is, alternative programmable effectors and systems for modifying nucleic acids and polynucleotides (i.e., DNA, RNA, or any hybrids, derivatives, or modifications thereof).

[0005] Summary of the Invention

[0006] On the one hand, the present application provides an isolated Cas enzyme, the Cas enzyme comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-58 or a sequence having at least about 80% identity to the amino acid sequence as shown in any one of the SEQ ID NOs: 1-58. For example, the Cas enzyme comprises a sequence having about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to the amino acid sequence as shown in any one of the SEQ ID NOs: 1-58. For example, the present application provides a Cas enzyme comprising an amino acid sequence as shown in the following table:

[0007] In certain embodiments, the Cas enzyme has a catalytically active domain capable of binding to a target DNA chain and / or a catalytically active domain capable of cleaving the target DNA chain.

[0008] In certain embodiments, the catalytically active domain comprises one or more amino acid changes, such that the Cas enzyme has only the activity of binding to the target DNA chain, or has the activity of binding to the target DNA chain and the activity of cutting the target DNA single chain.

[0009] On the other hand, the present application provides a fusion molecule, which comprises the Cas enzyme described in the present application and one or more heterologous functional domains.

[0010] In certain embodiments, the one or more heterologous functional domains are capable of regulating the expression of one or more gene products.

[0011] In certain embodiments, the one or more heterologous functional domains are fused directly or indirectly to the Cas enzyme.

[0012] In certain embodiments, the one or more heterologous functional domains are selected from the group consisting of an autohelicase, a nuclease, a helicase-nuclease, a DNA methyltransferase, a DNA hydroxymethylase, a histone methylase, a histone demethylase, a histone acetyltransferase, a histone deacetylase, a phosphatase, a kinase, a transcription (co)activator, a transcription repressor, a DNA binding protein, a DNA structural protein, a marker protein, a reporter protein, a fluorescent protein, a ligand binding protein, a signal peptide, a subcellular localization sequence, an antibody epitope, and an affinity purification tag.

[0013] In certain embodiments, the one or more heterologous functional domains have one or more of the following activities: methylase activity, demethylase activity, deaminase activity, transcription activation activity, transcription repression activity, transcription release factor activity, reverse transcriptase activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity.

[0014] On the other hand, the present application provides an engineered, programmable, non-naturally occurring CRISPR-Cas system, which comprises the Cas enzyme described in the present application or the fusion molecule described in the present application, and one or more guide RNAs, wherein the one or more guide RNAs target the loci of nucleic acid molecules encoding one or more gene products in the cell, thereby guiding the Cas enzyme or the fusion molecule to bind to and / or cut the loci of the nucleic acid molecules encoding one or more gene products; and the Cas enzyme or the fusion molecule and the guide RNA do not naturally exist together.

[0015] On the other hand, the present application provides an engineered, non-naturally occurring vector system, comprising one or more vectors, the one or more vectors comprising: a) a first regulatory element, the first regulatory element being operably linked to one or more guide RNAs, the one or more guide RNAs being capable of hybridizing to a target sequence in a locus of a nucleic acid molecule encoding one or more gene products, and b) a second regulatory element, the second regulatory element being operably linked to the Cas enzyme described herein or the fusion molecule described herein, wherein the components a) and b) are located on the same or different vectors of the vector system, and the guide RNA targets the locus of the nucleic acid molecule encoding one or more gene products in the cell, thereby guiding the Cas enzyme or the fusion molecule to bind to and / or cut the locus of the nucleic acid molecule encoding one or more gene products; and the Cas enzyme or the fusion molecule does not naturally exist together with the guide RNA.

[0016] In certain embodiments, expression of the one or more gene products is altered.

[0017] In certain embodiments, expression of the gene product is decreased or increased.

[0018] In certain embodiments, the gene product is a protein.

[0019] In certain embodiments, the cell is a eukaryotic cell.

[0020] In certain embodiments, the eukaryotic cell is a mammalian cell. For example, the mammalian cell includes, but is not limited to, cells of mice, monkeys, humans, farm animals, sports animals, and pets.

[0021] In certain embodiments, the mammalian cell is a human cell.

[0022] In certain embodiments, the Cas enzyme is codon-optimized for expression in eukaryotic cells.

[0023] In certain embodiments, the guide RNA comprises a guide sequence fused to a tracr sequence.

[0024] In certain embodiments, the guide RNA comprises a direct repeat sequence and a spacer sequence, wherein the spacer sequence binds to a nucleic acid molecule targeted by the guide RNA.

[0025] In certain embodiments, the direct repeat sequence is 10 to 70 nucleotides in length.

[0026] In certain embodiments, the direct repeat sequence is 31 to 36 nucleotides in length.

[0027] In certain embodiments, the direct repeat sequence comprises the nucleotide sequence shown in any one of SEQ ID NOs: 63-88 and 90-99, or comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence shown in any one of SEQ ID NOs: 63-88 and 90-99.

[0028] In certain embodiments, the spacer sequence is 16 to 24 nucleotides in length.

[0029] In certain embodiments, the nucleic acid molecule targeted by the guide RNA comprises a nucleotide sequence capable of complementary pairing with the spacer sequence.

[0030] In certain embodiments, the vector or the Cas enzyme of the system further comprises one or more nuclear localization sequences (NLS).

[0031] In certain embodiments, the system is introduced into the cell via a delivery system selected from the group consisting of viral particles, liposomes, lipid nanoparticles, electroporation, microinjection, and conjugation.

[0032] On the other hand, the present application provides a method for altering the expression of one or more gene products, the method comprising introducing an engineered, non-naturally occurring CRISPR-Cas system into a cell comprising and expressing a nucleic acid molecule encoding the one or more gene products, the system comprising the Cas enzyme described herein or the fusion molecule described herein, and one or more guide RNAs, the one or more guide RNAs targeting the locus of the nucleic acid molecule encoding the one or more gene products, thereby guiding the Cas enzyme or the fusion molecule to bind to and / or cut the locus, thereby altering the expression of the one or more gene products; and the Cas enzyme or the fusion molecule and the guide RNA do not naturally exist together.

[0033] On the other hand, the present application provides a method for altering the expression of one or more gene products, the method comprising introducing an engineered, non-naturally occurring vector system into a cell containing and expressing a nucleic acid molecule encoding the one or more gene products, the vector system comprising one or more vectors, the one or more vectors comprising: a) a first regulatory element, the first regulatory element being operably linked to one or more guide RNAs, the one or more guide RNAs being capable of hybridizing to a target sequence in the locus of the nucleic acid molecule encoding the one or more gene products, and b) a second regulatory element, the second regulatory element being operably linked to the Cas enzyme described herein or the fusion molecule described herein, wherein the components a) and b) are located on the same or different vectors of the vector system, and the guide RNA targets the locus of the nucleic acid molecule encoding the one or more gene products in the cell, thereby guiding the Cas enzyme or the fusion molecule to bind to and / or cut the locus, thereby altering the expression of the one or more gene products; and the Cas enzyme or the fusion molecule does not naturally exist together with the guide RNA.

[0034] In certain embodiments, expression of the gene product is decreased or increased.

[0035] In certain embodiments, the gene product is a protein.

[0036] In certain embodiments, the cell is a eukaryotic cell.

[0037] In certain embodiments, the eukaryotic cell is a mammalian cell. For example, the mammalian cell includes, but is not limited to, cells of mice, monkeys, humans, farm animals, sports animals, and pets.

[0038] In certain embodiments, the mammalian cell is a human cell.

[0039] In certain embodiments, the Cas enzyme is codon-optimized for expression in eukaryotic cells.

[0040] In certain embodiments, the guide RNA comprises a guide sequence fused to a tracr sequence.

[0041] In certain embodiments, the guide RNA comprises a direct repeat sequence and a spacer sequence, wherein the spacer sequence binds to a nucleic acid molecule targeted by the guide RNA.

[0042] In certain embodiments, the direct repeat sequence is 10 to 70 nucleotides in length.

[0043] In certain embodiments, the direct repeat sequence is 31 to 36 nucleotides in length.

[0044] In certain embodiments, the direct repeat sequence comprises the nucleotide sequence shown in any one of SEQ ID NOs: 63-88 and 90-99, or comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence shown in any one of SEQ ID NOs: 63-88 and 90-99.

[0045] In certain embodiments, the spacer sequence is 16 to 24 nucleotides in length.

[0046] In certain embodiments, the nucleic acid molecule targeted by the guide RNA comprises a nucleotide sequence capable of complementary pairing with the spacer sequence.

[0047] In certain embodiments, the vector or the Cas enzyme of the system further comprises one or more nuclear localization sequences (NLS).

[0048] In certain embodiments, the method comprises introducing the CRISPR-Cas system or the vector system into the cell via a delivery system selected from the group consisting of viral particles, liposomes, lipid nanoparticles, electroporation, microinjection, and conjugation.

[0049] On the other hand, the present application provides a nucleic acid encoding the Cas enzyme described in the present application, the fusion molecule described in the present application, or the CRISPR-Cas system described in the present application.

[0050] On the other hand, the present application provides a cell comprising the Cas enzyme described herein, the fusion molecule described herein, the CRISPR-Cas system described herein, the vector system described herein and / or the nucleic acid described herein.

[0051] On the other hand, the present application provides a kit comprising the Cas enzyme described herein, the fusion molecule described herein, the CRISPR-Cas system described herein, the vector system described herein, the nucleic acid described herein and / or the cell described herein.

[0052] In certain embodiments, the kit further comprises a container for placing the Cas enzyme, the fusion molecule, the CRISPR-Cas system, the vector system, the nucleic acid and / or the cell, and instructions for use.

[0053] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0055] Figures 1A-1B show a schematic diagram of the assay process for the cleavage activity of the Cas enzyme described in the present application in eukaryotic cells, the fluorescence results, and the PAM motif required for gRNA binding of the Cas enzyme.

[0056] Figures 2A-2B show a schematic diagram of the determination process of the cleavage activity of the Cas enzyme described in this application in eukaryotic cells and the comparison results of green fluorescent cells of different Cas enzymes.

[0057] Figures 3A-3B show the schematic diagram of the determination process of the in vitro cleavage activity of the Cas enzyme described in this application and its PAM sequence and the sequencing results.

[0058] Figure 4 shows the site information and amplification detection results of detecting the Cas enzyme activity described in this application at the endogenous site.

[0059] Figures 5A-5C show the GFP fluorescence results of different variants of the Cas enzyme described in this application after cleavage activity assay in eukaryotic cells.

[0060] Figures 6A-6B show the results of the green fluorescent cell ratio after the cleavage activity of sgRNA treated with the Cas enzyme described in this application combined with different DR (Direct repeat) region optimization schemes in eukaryotic cells was measured.

[0061] Figure 7 shows the results of the green fluorescent cell ratio after the cleavage activity of the Cas enzyme described in this application combined with sgRNA of different Spacer lengths in eukaryotic cells was measured.

[0062] Figures 8A-8B show the results of green fluorescent cell ratios after cleavage activity assays of different variants of the Cas enzyme described in the present application in eukaryotic cells. Figure 8C shows the results of cleavage activity assays (green fluorescent cell ratios) of different variants of the Cas enzyme described in the present application in different PAM reporter systems.

[0063] Figures 9A-9C show a schematic diagram of the structure of a cytosine base editor comprising the Cas enzyme described in this application, as well as the results of detecting its base editing efficiency at two endogenous sites, EMX1 and VEGFA.

[0064] FIG10 shows the activation effect of the gene activation epigenetic tool comprising the Cas enzyme described in the present application on detecting target site gene expression at the CXCR4 endogenous site. DETAILED DESCRIPTION

[0065] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0066] Definition of terms

[0067] In the present application, the term "identity" can be used interchangeably with "homology", and it generally refers to the relationship between two or more polypeptide molecules or two or more nucleic acid molecule sequences, and this relationship is determined by comparing their sequences. In the art, "identity" also refers to the degree of nucleic acid molecules or polypeptide sequence dependency, which can be determined by the coupling between two or more nucleotides or two or more amino acid residues. In the present application, the identity percentage (%) of an amino acid sequence is defined as the percentage of the total number of residues in the candidate sequence that is identical with the amino acid residue in the reference polypeptide sequence after comparing sequences and, if necessary, introducing a gap to reach maximum percentage sequence identity, and not considering any conservative substitution as part of sequence identity. The comparison for the purpose of measuring percentage amino acid sequence identity can be reached in a variety of ways within the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for comparing sequences, including any algorithm required for maximum comparison within the full length of the sequence being compared. In certain embodiments, the calculation of the percent identity (%) of a polypeptide molecule or nucleic acid molecule sequence can also determine the total number of residues based on the type of sequence mutation. Mutation types include insertions (extensions) at either or both ends of the sequence, deletions (truncations) at either or both ends of the sequence, substitutions / alternations of one or more amino acids / nucleotides, insertions within the sequence, and deletions within the sequence. Taking the amino acid sequence of a polypeptide as an example (the same applies to nucleotide sequences), if the mutation type is one or more of the following: substitutions / alternations of one or more amino acids / nucleotides, insertions within the sequence, and deletions within the sequence, the total number of residues is calculated based on the larger of the two compared molecules. If the mutation type also includes insertions (extensions) at either or both ends of the sequence, deletions (truncations) at either or both ends of the sequence, or insertions within the sequence and deletions within the sequence, the number of amino acids inserted or deleted at either or both ends or within the sequence (e.g., the number of insertions or deletions at both ends is less than 20) is not counted in the total number of residues. When calculating the percent identity, the sequences being compared can be aligned in a manner that produces the maximum match between the sequences, and gaps in the alignment (if any) are resolved by a specific algorithm.

[0068] In the present application, "catalytically active domain" refers to an identifiable or determinable conserved structural entity within the Cas protein (enzyme) that exhibits significant secondary structure content, and the conserved structure is a region where the Cas protein (enzyme) performs functions such as binding and / or cleavage of polynucleotides. An exemplary catalytically active domain can be a protease of the Cas9 family, which has two catalytically active domains, one of which is HNH-like, and its function is to cut off the single-stranded polynucleotide (target chain) paired with the guide RNA, and the other domain is RuvC-like, and its function is to cut off the complementary chain of the target chain.

[0069] In this application, the term "binding" (e.g., with respect to a target DNA binding (catalytically active) domain of a polypeptide or protease) generally refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). When in a state of non-covalent interaction, the macromolecules are said to be "associated" or "interacting" or "bound" (e.g., when molecule X is said to interact with molecule Y, it means that molecule X binds to molecule Y in a non-covalent manner). It will be understood that not all components of the binding interaction need to be sequence-specific (e.g., contacts with phosphate residues in the DNA backbone), but some portions of the binding interaction may be sequence-specific.

[0070] In the application, the term "fusion molecule" generally refers to a molecule (bipartite molecule) consisting of at least two parts, which includes an enzyme (protein or peptide) of the present application, which is coupled to at least one other part to form a single entity. The enzyme and the at least one other part can be separated by a joint, or can be directly coupled. The at least one other part can be fused to the enzyme of the present application at any amino acid outside the N-terminal, C-terminal or terminal amino acid. The other parts can be fused to the part already included in the fusion molecule. Those skilled in the art fully understand the best order and / or combination of the determination of the parts in the fusion molecule of the present application. Usually, when the fusion molecule includes an enzyme of the present application and at least one other peptide, the term does not include such a fusion molecule in which the fusion produces a naturally occurring peptide.

[0071] In the present application, the term "heterologous" generally refers to a nucleotide or polypeptide sequence that is not present in a natural nucleic acid or protein, respectively. In certain embodiments of the present application, the term "heterologous functional domain" may refer to about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains or a part of a fusion molecule described in the present application in addition to the Cas enzyme described in the present application. Examples of heterologous functional domains that may be included in the fusion molecule described in the present application or that may be fused to the Cas enzyme described in the present application include, but are not limited to, epitope tags, reporter gene sequences, and one or more protein domains having the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza virus hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. The example of reporter gene includes, but is not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), to include the autofluorescent protein of blue fluorescent protein (BFP). Cas enzyme can be fused to the gene sequence encoding a kind of protein or protein fragment, and described protein or protein fragment binds DNA molecule or binds other cellular molecules, it includes, but is not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusion, GAL4DNA binding domain fusion and herpes simplex virus (HSV) BP16 protein fusion. Other domains that can form a part of the fusion molecule comprising Cas enzyme are described in US20110059502, which is incorporated herein by reference.

[0072] In this application, the term "expression" generally refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0073] In this application, the terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They generally refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, multiple loci (a locus) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be performed before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeled component.

[0074] In this application, the terms "non-naturally occurring" and "engineered" are used interchangeably. When referring to nucleic acid molecules, polypeptides, or combinations and systems thereof, they generally mean that the nucleic acid molecules or polypeptides are at least substantially free from at least one other component with which they are associated in nature or as found in nature.

[0075] In this application, the term "vector" generally refers to a nucleic acid molecule that is capable of transporting another nucleic acid molecule connected thereto. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, no free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and other diverse polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which another DNA fragment can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which a virally derived DNA or RNA sequence is present in a vector for packaging a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an adeno-associated virus). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) are capable of autonomous replication in the host cell into which they are introduced. Other vectors (e.g., non-additional mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell and are thereby replicated together with the host genome. In another embodiment, the present invention relates to a nucleic acid sequence that is operably connected to a host cell. In another embodiment, the present invention relates to a nucleic acid sequence that is operably connected to a host cell. In another embodiment, the present invention relates to a nucleic acid sequence that is operably connected to a host cell. In another embodiment, the present invention relates to a nucleic acid sequence that is operably connected to a host cell. In another embodiment, the present invention relates to a nucleic acid sequence that is operably connected to a host cell. In another embodiment, the present invention relates to a nucleic acid sequence that is operably connected to a host cell. In another embodiment, the present invention relates to a nucleic acid sequence that is operably connected to a host cell. In another embodiment, the present invention relates to a nucleic acid sequence that is operably connected to a host cell. In another embodiment, the present invention relates to a nucleic acid sequence that is operably connected to a host cell.

[0076] In this application, the term "regulatory element" is generally intended to include promoters, enhancers, internal ribosome entry sites (IRES) and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory sequences are, for example, described in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California (1990). In certain embodiments, regulatory elements may include those sequences that direct the constitutive expression of a nucleotide sequence in many types of host cells and those sequences (e.g., tissue-specific regulatory sequences) that direct the nucleotide sequence to be expressed only in certain host cells. Tissue-specific promoters can primarily direct expression in desired tissues of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or special cell types (e.g., lymphocytes). Regulatory elements can also direct expression in a time-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue- or cell-type-specific. In certain embodiments, a vector may comprise one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or a combination thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter, and the EF1α promoter. The term "regulatory element" may also encompass enhancer elements such as WPRE, CMV enhancer, R-U5' fragment in the LTR of HTLV-I, SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

[0077] Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the desired expression level, etc. A vector can be introduced into a host cell to thereby produce transcripts, proteins, or peptides, including fusion molecules or enzymes encoded by nucleic acids as described herein (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion molecules or fusion proteins thereof, etc.). Advantageous vectors include lentiviruses and adeno-associated viruses, and vectors of this type may also be selected to target specific cell types.

[0078] In this application, the term "codon optimization" generally refers to a method of modifying a nucleic acid sequence to enhance expression in a host cell by replacing at least one codon of a native sequence with a codon that is more frequently or most frequently used in the genes of a host cell, such as approximately or more than approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons while maintaining the native amino acid sequence. Different species exhibit specific preferences for certain codons with specific amino acids. Codon preference (the difference in codon usage between organisms) is often associated with the translation efficiency of messenger RNA (mRNA), and this translation efficiency is considered to depend, among other things, on the properties of the codons translated and the availability of specific transfer RNA (tRNA) molecules. The advantage of the tRNA selected in the cell generally reflects the codons that are most frequently used for peptide synthesis. Therefore, genes can be customized to the optimal gene expression based on codon optimization in a given organism. Codon utilization tables can be easily obtained, such as in the Codon Usage Database, and these tables can be adjusted and applied in different ways. For example, see Nakamura Y. et al., Codon usage tabulated from the international DNA sequence databases: status for the year 2000, "Nucl. Acids Res." 28: 292 (2000). Computer algorithms for codon optimization of specific sequences for expression in specific host cells are also available, such as Gene Forge (Aptagen, Jacobus, PA). In certain embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in the sequence encoding the Cas enzyme correspond to the most frequently used codons for a particular amino acid.

[0079] In this application, the term "guide RNA" is used interchangeably with "guide RNA" and "gRNA", and generally refers to a group of nucleic acid molecules that facilitate the specific guidance of RNA-guided nucleases or other effector molecules (usually complexed with gRNA molecules) to a target sequence. In nature, crRNA and tracrRNA generally exist as two independent RNA molecules, constituting gRNA. The term "tracrRNA" generally refers to a scaffold-type RNA that can bind to the Cas nuclease, and the term "crRNA" is also called CRISPR RNA, which generally refers to a nucleotide sequence that is complementary to the target DNA being targeted. CrRNA and tracRNA can also be fused into a single strand, in which case the gRNA can also be called a single-stranded guide RNA (sgRNA). sgRNA has become the most common form of gRNA used by those skilled in the art in CRISPR technology, so the terms "sgRNA" and "gRNA" can have the same meaning in this article. sgRNA can be artificially synthesized or prepared from a DNA template in vitro or in vivo. sgRNA can bind to the Cas nuclease or target the target DNA, and it can guide the Cas nuclease to cut the DNA site complementary to the gRNA.

[0080] As used herein, crRNA generally comprises a spacer sequence that mediates target recognition and a direct repeat sequence (also referred to herein as "Direct repeat" or "DR sequence") that forms a complex with the CRISPR-Cas effector protein. In some cases, the spacer sequence (also known as a guide sequence) is any polynucleotide sequence that has sufficient complementarity with the target sequence to hybridize with the target sequence and guide the specific binding of the CRISPR-Cas system complex to the target sequence. In certain embodiments, when optimally aligned, the degree of complementarity between the spacer sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. Determining the optimal alignment is within the capabilities of those of ordinary skill in the art. For example, there are publicly available and commercially available alignment algorithms and programs, such as, but not limited to, ClustalW, the Smith-Waterman algorithm in Matlab, Bowtie, Geneious, Biopython, and SeqMan.

[0081] In certain embodiments, the spacer sequence is at least 5, at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides in length. In certain embodiments, the spacer sequence is no more than 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 15, 10, or less nucleotides in length. In certain embodiments, the spacer sequence is 10-30, 15-25, 15-22, 16-24, 19-25, or 19-22 nucleotides in length. In certain preferred embodiments, the spacer sequence is 20 nucleotides in length.

[0082] In certain embodiments, the direct repeat sequence is at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 40, at least 45, at least 50, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, or at least 70 nucleotides in length. In certain embodiments, the direct repeat sequence is no more than 70, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 15, 10 or fewer nucleotides in length. In certain embodiments, the direct repeat sequence is 55-70 nucleotides in length, such as 55-65 nucleotides, such as 60-65 nucleotides, such as 62-65 nucleotides, such as 63-64 nucleotides. In certain embodiments, the direct repeat sequence is 15-40 nucleotides, such as 15-25 nucleotides, such as 20-30 nucleotides, such as 22-36 nucleotides, such as 31 nucleotides in length.

[0083] In this application, the terms "comprise" and "include" are used interchangeably, which generally refer to including the features specified but not excluding other elements. The term "at least" generally refers to including the number specified.

[0084] In this application, the term "about" or "approximately" generally refers to an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "approximately" can mean within 1 or more than 1 standard deviation, as practiced in the art. Alternatively, "about" or "approximately" can mean a range of up to 10% or 20% (i.e., ±10% or ±20%).

[0085] In this application, the term "selected from" generally refers to the selected objects and all combinations thereof. For example, "selected from (:) A, B and C" means all combinations of A, B and C, for example, A, B, C, A+B, A+C, B+C or A+B+C.

[0086] Without intending to be bound by any theory, the following examples are merely intended to illustrate the enzyme and its fusion molecules, methods and uses of the present application, and are not intended to limit the scope of the present invention.

[0087] Example

[0088] Example 1

[0089] Determination of the cleavage activity of the Cas enzyme of the present application in eukaryotic cells

[0090] This example uses stably transfected 293T cells with a blue-green light reporter system to test the activity of the Cas enzyme provided by this application. As shown in Figure 1, the reporter system contains a continuously expressed CMV promoter, a sequence encoding blue fluorescent protein, a sequence encoding green fluorescent protein, and a gRNA targeting sequence inserted in the middle; the gRNA targeting sequence is flanked by random N-base sequences, which can be used to screen for Cas enzymes with different protospacer adjacent motif (PAM) preferences.

[0091] The Cas enzyme cleavage activity of the present application will be determined by the fluorescence results revealed by the reporter system, and the reporter system in which no cleavage occurs will stably express blue fluorescent protein to emit blue fluorescence, and the sequence encoding green fluorescent protein is not expressed due to the presence of a stop codon, so that green fluorescent protein cannot be expressed, and the reporter system will not emit green fluorescence. Only after DNA cleavage occurs near the gRNA targeting sequence, and frameshift mutation occurs in the cell during the repair of the incision, can green fluorescent protein be expressed while blue fluorescent protein is stably expressed. The fluorescence results of Figure 1A show that after transfection of the Cas enzyme (SEQ ID NO: 2) provided in the present application, the experimental group cells with cleavage activity will produce a cell group that clearly expresses green fluorescence.

[0092] The green fluorescent cell population is enriched by flow cytometry, the target region is amplified after genome extraction, and the PAM motif required for gRNA binding of the Cas enzyme provided in this application can be determined by high-throughput sequencing (as shown in Figure 1B).

[0093] In this example, an exemplary nucleotide sequence encoding a reporter system is shown below (blue and green fluorescent protein sequences in bold, linker sequences in italics, 2A cleavage peptide sequences in bold and underlined, and an exemplary gRNA targeting sequence containing random N bases (SEQ ID NO: 59)):

[0094] Example 2

[0095] Determine the cleavage activity of the Cas enzyme of the present application in eukaryotic cells

[0096] The Cas enzyme provided in this application (SEQ ID NOs: 2, 47-51 and 54), the corresponding gRNA and the reporter plasmid with the gRNA targeting site are co-transfected into HEK293T cells. By transforming the targeting sequence on the reporter plasmid, the reporter system required for different Cas enzymes can be quickly constructed. As shown in Figure 2A, the reporter system cells in which no cutting occurs only express blue fluorescent protein, and the green fluorescent protein cannot be expressed normally because it is cut off. At this time, the reporter system only emits blue fluorescence. After the gRNA targeting site is cut, the cell repairs the DNA by homologous recombination to obtain a complete green fluorescent protein expression frame, so that the reporter system emits green fluorescence.

[0097] Cas enzymes with stronger cleavage activity will produce a higher proportion of DNA cleavage, thereby obtaining more copies of repaired green fluorescent protein, and the cells will show stronger green fluorescence. Therefore, this example quickly compares the cleavage activity of different Cas enzymes by comparing the proportion and fluorescence intensity of green fluorescent cells. The comparison results are shown in Figure 2B.

[0098] In this example, an exemplary nucleotide sequence encoding a reporter system is shown below (blue and green fluorescent protein sequences are in bold, the linker sequence is in italics, and the 2A cleavage peptide sequence is underlined; the exemplary gRNA targeting sequence (SEQ ID NO: 60) is in bold and underlined, wherein the TTTG and TGG at both ends of the sequence will be set to different sequences according to the PAM preference of different Cas enzymes):

[0099] Example 3

[0100] Determination of the in vitro cleavage activity of the Cas enzyme of the present application and its PAM sequence

[0101] As shown in the experimental design flow chart of this embodiment in Figure 3A, the Cas enzyme provided by the present application is expressed and purified in Escherichia coli, and reacted with a plasmid library containing a PAM library together with an in vitro transcribed gRNA (the gRNA targeting sequence is SEQ ID NO: 59, and it has random N base sequences on both sides for determining PAM preference). The Cas enzyme with cutting activity will cut the plasmid library to form linearized DNA. Through in vitro ligation, the adapter fragment is connected to the library fragment after DNA linearization, and then the vector and the library fragment connected to the adapter are amplified by specific PCR primers and high-throughput sequencing is performed.

[0102] The analysis and sequencing results shown in Figure 3B show the preference of Cas enzymes with cleavage activity for PAM motifs. These results show that the four proteins EpiCas037 (SEQ ID NO: 36), EpiCas044 (SEQ ID NO: 43), EpiCas040 (SEQ ID NO: 39) and EpiCas045 (SEQ ID NO: 44) all have cleavage activity and prefer T-rich PAM motifs.

[0103] Example 4

[0104] Detection of Cas enzyme activity of the present application at endogenous sites

[0105] According to the PAM motifs of different Cas enzymes determined in Example 3, qualified sites were found in the endogenous sites of HEK293T, and gRNA was constructed (gRNA targeting sequence was SEQ ID NO: 89). The Cas enzyme (SEQ ID NOs: 44) provided by the present application and the corresponding gRNA were co-transfected into HEK293T cells. After 3 days of transfection, the transfected positive cells were enriched by flow sorting, and the target region was amplified after the cells were lysed. The amplified PCR fragments were reacted with T7 endonuclease. If the Cas enzyme cuts the target site, various random repair sequences will be formed, which will be cut by the T7 enzyme to produce bands other than the target band. Therefore, the results shown in Figure 4 indicate that the Cas enzyme has cutting activity at the endogenous site.

[0106] Example 5

[0107] Determine the cleavage activity of the Cas enzyme mutants of the present application in eukaryotic cells

[0108] By site-directed mutagenesis, the Cas enzyme (SEQ ID NO:2) provided by the application is mutated to target amino acid (see the table below) at different sites, and then the detection as described in Example 2 is carried out. Wherein, DR (direct repeat) regional sequence of gRNA and Spacer sequence are respectively as shown in SEQ ID NO:64 and 60. The result shown in Fig. 5 A shows that m1, m2, m4, m5, m7, m11, m15 mutant activity are significantly improved compared to wild type. After these effective mutations are superimposed, the activity detection of the same method is carried out. The result shown in Fig. 5 B shows that the double mutant formed by the mutation superposition of two kinds selected from the table below can further enhance activity, and the activity of some double mutants is suitable for AsCas12a;The result shown in Fig. 5 C shows that the triple mutant and quadruple mutant formed by further superimposing effective single mutation and double mutation still maintain original activity, and activity is suitable for AsCas12a. The amino acid sequence of AsCas12a for comparison is as shown in SEQ ID NO:127.

[0109] Example 6

[0110] Optimization of CRISPR / Cas editing systems

[0111] This example optimizes the editing system comprising the Cas enzyme (SEQ ID NO: 57) and gRNA provided herein. The Cas enzyme activity was detected using the reporter system and detection method described in Example 2.

[0112] gRNA optimization:

[0113] The DR (direct repeat) region of gRNA was changed to 36nt (SEQ ID NO:87), 31nt (SEQ ID NO:91), and 22nt (SEQ ID NO:90) by molecular cloning. The results shown in Figure 6A show that a DR of 31nt length can maintain 100% Cas enzyme activity. When the base pairs in the DR region are changed by molecular cloning to improve its stability (when DR is 31.1 to 31.8, its nucleotide sequence is shown in SEQ ID NO:92-99), the results shown in Figure 6B show that most of the changes have little effect on the activity, while the change of 31.8 can most preferably enhance the activity. This embodiment also changes the length of the spacer region (Spacer) for targeting DNA by molecular cloning to improve its binding efficiency, thereby enhancing the activity of the Cas enzyme. As shown in Figure 7, when the Spacer length changes from 24nt to 16nt (SEQ ID NO:100-103, 60 and 104-107), the Cas enzyme provided in this application can maintain activity.

[0114] Cas enzyme mutants:

[0115] By site-directed mutagenesis, different sites of the Cas enzyme (SEQ ID NO: 57) provided herein were mutated into target amino acids (see the table below), and then detected as described in Example 2. Wherein, the DR (direct repeat) region sequence and the Spacer sequence of the gRNA are shown in SEQ ID NOs: 91 and 60, respectively. The results shown in Figures 8A-8B show that most single mutations have little effect on the activity of the Cas enzyme. If the single mutations with better activity optimization effects are superimposed, it can be found that the double mutants formed can still maintain high activity.

[0116] [Corrected 03.12.2024 according to Rule 91] In this example, based on the reporter system described in Example 2, TTTG was mutated into TTTH, TTVG, TVTG and VTTG (H represents A / C / T, V represents A / C / G, and the three plasmids were mixed in equal proportions and transfected) to construct a new PAM reporter system. The results shown in Figure 8C showed that some mutants were able to broaden the range of PAM recognition, especially in the recognition of PAM targets of VTTG and TTVG. Compared with the wild type, the Cas enzyme activity provided in this application was significantly improved.

[0117] Example 7

[0118] Detecting the effects of inactivated Cas enzymes (dCas) at endogenous loci

[0119] Base editing effect of dCas-CBE:

[0120] The Cas enzyme provided in the present application (SEQ ID NO: 57) was inactivated and then fused with rAPOBEC1 and UGI ( FIG. 9A ) to obtain a cytosine base editor based on the Cas enzyme provided in the present application. The cytosine base editor was co-transfected into HEK293T cells with the corresponding gRNA (the DR sequence is shown in SEQ ID NO: 91, and the Spacer sequences targeting the EMX1 site and the VEGFA site are shown in SEQ ID NOs: 108 and 109, respectively). After 48 hours of transfection, the transfected cells were sorted, the genome was extracted, the target site was amplified, and the base editing efficiency was determined by Sanger sequencing. The results are shown in FIG. 9B-9C .

[0121] The amino acid sequence of the dEpiCas059m46-CBE editor is shown below (SEQ ID NO: 116, italic NLS, italic bold rAPOBEC1, bold dEpiCas059, italic underlined UGI, underlined P2A cleavage peptide, and <> is the mCherry marker; its plasmid sequence is SEQ ID NO: 117):

[0122] The amino acid sequence of the dAsCas12a-CBE editor is shown below (SEQ ID NO: 118, italic NLS, italic bold rAPOBEC1, bold dAsCas12a, italic underlined UGI, underlined P2A cleavage peptide, and <> is the mCherry marker; its plasmid sequence is SEQ ID NO: 119):

[0123] Gene activation effects of dCas-SPH:

[0124] The Cas enzyme (SEQ ID NO: 55) provided in this application was inactivated and then fused with 10×GCN4 to recruit the scFV-P65-HSF1 fusion peptide, thereby obtaining a gene activation tool based on the Cas enzyme provided in this application. The principle of this tool is based on the ability of GCN4 to spontaneously recognize and bind to scFV, thereby enriching the P65 and HSF1 effectors with transcriptional activation function near the target site of the Cas enzyme, thereby activating gene expression at the target site.

[0125] The transcriptional activation tool and the gRNA targeting CXCR4 (Spacer sequence is shown in SEQ ID NOs: 120-123, and the four gRNAs are mixed in equal proportions) were co-transfected into HEK293T cells. After 48 hours of transfection, the cells were collected and stained with PE anti-human CXCR4 antibody (BioLegendg, 306506). The fluorescence intensity of the PE channel was detected by flow cytometry to reflect the expression intensity of CXCR4. The activation efficiency (MFI fold change) was obtained by dividing the PE mean fluorescence intensity of the transfection-positive group by the PE mean fluorescence intensity of the transfection-negative group. It is used to represent the activation intensity of different activation tools (Figure 10), and then represents the DNA binding effect of different tools.

[0126] The amino acid sequence of the scFV-P65-HSF1 fusion peptide is shown below (SEQ ID NO: 124, italics indicates NLS, italic bold indicates P65 and HSF1, bold indicates scFV, italics underline indicates HA tag, underline indicates linker peptide, and <> indicates Flag tag):

[0127] The amino acid sequence of the dEpiCas057-10×GCN4 fusion peptide is shown below (SEQ ID NO: 125, NLS in italics, dEpiCas057 in bold, and GCN4 in <>):

[0128] The fusion peptide scFV-P65-HSF1 and dEpiCas057-10×GCN4 can be expressed together by the plasmid sequence shown in SEQ ID NO:126.

[0129] The above experiments demonstrate that the Cas enzyme provided in this application, which loses its cleavage activity through mutation, is suitable for base editing and epigenetic modification editing applications, and is not limited to other DNA-targeted application scenarios such as gene activation, gene silencing, chromosome imaging, and the like.

Claims

1. An isolated Cas enzyme, comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-58 or a sequence having at least about 80% identity with the amino acid sequence as shown in any one of SEQ ID NOs: 1-58.

2. The Cas enzyme according to claim 1, wherein the Cas enzyme has a catalytically active domain capable of binding to a target DNA chain and / or a catalytically active domain capable of cutting the target DNA chain.

3. According to the Cas enzyme according to any one of claims 1-2, the catalytic activity domain comprises one or more amino acid changes, so that the Cas enzyme has only the activity of binding to the target DNA chain, or has the activity of binding to the target DNA chain and the activity of cutting the target DNA single strand.

4. A fusion molecule comprising the Cas enzyme described in any one of claims 1-3 and one or more heterologous functional domains.

5. The fusion molecule according to claim 4, wherein the one or more heterologous functional domains are capable of regulating the expression of one or more gene products.

6. According to the fusion molecule described in any one of claims 4-5, the one or more heterologous functional domains are directly or indirectly fused to the Cas enzyme.

7. according to the fusion molecule described in any one in claim 4-6, described one or more heterologous functional domains are selected from autohelicase, nuclease, helicase-nuclease, DNA methyltransferase, DNA hydroxymethylase, histone methylase, histone demethylase, histone acetyltransferase, histone deacetylase, phosphatase, kinase, transcription (total) activator, transcription repressor, DNA binding protein, DNA structural protein, marker protein, reporter protein, fluorescent protein, ligand binding protein, signal peptide, subcellular localization sequence, antibody epitope and affinity purification tag.

8. According to the fusion molecule described in any one of claims 4-7, the one or more heterologous functional domains have one or more of the following activities: methylase activity, demethylase activity, deaminase activity, transcription activation activity, transcription repression activity, transcription release factor activity, reverse transcriptase activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.

9. An engineered, programmable, non-naturally occurring CRISPR-Cas system, the system comprising the Cas enzyme of any one of claims 1-3 or the fusion molecule of any one of claims 4-8, and one or more guide RNAs, wherein the one or more guide RNAs target the loci of nucleic acid molecules encoding one or more gene products in a cell, thereby guiding the Cas enzyme or the fusion molecule to bind to and / or cut the loci of the nucleic acid molecules encoding one or more gene products; and the Cas enzyme or the fusion molecule does not naturally exist together with the guide RNA.

10. An engineered, non-naturally occurring vector system, comprising one or more vectors comprising: a) a first regulatory element operably linked to one or more guide RNAs capable of hybridizing to a target sequence in a locus of a nucleic acid molecule encoding one or more gene products, and b) a second regulatory element, which is operably linked to the Cas enzyme of any one of claims 1 to 3 or the fusion molecule of any one of claims 4 to 8, Wherein, the component a) and the component b) are located on the same or different vectors of the vector system, and the guide RNA targets the locus of the nucleic acid molecule encoding one or more gene products in the cell, thereby guiding the Cas enzyme or the fusion molecule to bind to and / or cut the locus of the nucleic acid molecule encoding one or more gene products; and the Cas enzyme or the fusion molecule does not naturally exist together with the guide RNA.

11. The system of any one of claims 9-10, wherein the expression of the one or more gene products is altered.

12. The system according to any one of claims 9 to 11, wherein the expression of the gene product is decreased or increased.

13. The system of any one of claims 9-12, wherein the gene product is a protein.

14. The system according to any one of claims 9 to 13, wherein the cell is a eukaryotic cell.

15. The system according to any one of claims 9 to 14, wherein the eukaryotic cell is a mammalian cell.

16. The system of any one of claims 9-15, wherein the mammalian cells are human cells.

17. The system of any one of claims 9-16, wherein the Cas enzyme is codon-optimized for expression in eukaryotic cells.

18. The system of any one of claims 9-17, wherein the guide RNA comprises a guide sequence fused to a tracr sequence.

19. The system according to any one of claims 9 to 18, wherein the guide RNA comprises a direct repeat sequence and a spacer sequence, wherein the spacer sequence binds to the nucleic acid molecule targeted by the guide RNA.

20. The system of claim 19, wherein the direct repeat sequence is 10 to 70 nucleotides in length.

21. The system of claim 19 or 20, wherein the direct repeat sequence is 31 to 36 nucleotides in length.

22. The system according to any one of claims 19-21, wherein the direct repeat sequence comprises the nucleotide sequence shown in any one of SEQ ID NOs: 63-88 and 90-99, or comprises a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence shown in any one of SEQ ID NOs: 63-88 and 90-99.

23. The system of claim 19, wherein the spacer sequence has a length of 16 to 24 nucleotides.

24. The system of claim 19 or 23, wherein the nucleic acid molecule targeted by the guide RNA comprises a nucleotide sequence capable of complementary pairing with the spacer sequence.

25. The system according to any one of claims 9-24, wherein the vector or the Cas enzyme of the system further comprises one or more nuclear localization sequences (NLS).

26. The system according to any one of claims 9-25, wherein the system is introduced into the cell via a delivery system selected from the group consisting of viral particles, liposomes, lipid nanoparticles, electroporation, microinjection and conjugation.

27. A method for altering the expression of one or more gene products, the method comprising introducing an engineered, non-naturally occurring CRISPR-Cas system into a cell containing and expressing a nucleic acid molecule encoding the one or more gene products, the system comprising the Cas enzyme of any one of claims 1-3 or the fusion molecule of any one of claims 4-8, and one or more guide RNAs, the one or more guide RNAs targeting the locus of the nucleic acid molecule encoding the one or more gene products, thereby guiding the Cas enzyme or the fusion molecule to bind to and / or cut the locus, thereby altering the expression of the one or more gene products; and the Cas enzyme or the fusion molecule does not naturally exist together with the guide RNA.

28. A method for altering the expression of one or more gene products, the method comprising introducing into a cell containing and expressing a nucleic acid molecule encoding the one or more gene products an engineered, non-naturally occurring vector system, the vector system comprising one or more vectors, the one or more vectors comprising: a) a first regulatory element operably linked to one or more guide RNAs capable of hybridizing to a target sequence in the locus of the nucleic acid molecule encoding one or more gene products, and b) a second regulatory element, which is operably linked to the Cas enzyme of any one of claims 1 to 3 or the fusion molecule of any one of claims 4 to 8, Wherein, the component a) and the component b) are located on the same or different vectors of the vector system, and the guide RNA targets the locus of the nucleic acid molecule encoding one or more gene products in the cell, thereby guiding the Cas enzyme or the fusion molecule to bind to and / or cut the locus, thereby changing the expression of the one or more gene products; and the Cas enzyme or the fusion molecule does not naturally exist together with the guide RNA.

29. The method of claim 27 or 28, wherein the expression of the gene product is decreased or increased.

30. The method of any one of claims 27-29, wherein the gene product is a protein.

31. The method of any one of claims 27-30, wherein the cell is a eukaryotic cell.

32. The method of any one of claims 27-31, wherein the eukaryotic cell is a mammalian cell.

33. The method of any one of claims 27-32, wherein the mammalian cell is a human cell.

34. The method of any one of claims 27-33, wherein the Cas enzyme is codon-optimized for expression in a eukaryotic cell.

35. The method of any one of claims 27-34, wherein the guide RNA comprises a guide sequence fused to a tracr sequence.

36. The method according to any one of claims 27-35, wherein the guide RNA comprises a direct repeat sequence and a spacer sequence, wherein the spacer sequence binds to the nucleic acid molecule targeted by the guide RNA.

37. The method of claim 36, wherein the direct repeat sequence is 10 to 70 nucleotides in length.

38. The method of claim 36 or 37, wherein the direct repeat sequence is 31 to 36 nucleotides in length.

39. The method according to any one of claims 36 to 38, wherein the direct repeat sequence comprises the nucleotide sequence shown in any one of SEQ ID NOs: 63 to 88 and 90 to 99, or comprises a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence shown in any one of SEQ ID NOs: 63 to 88 and 90 to 99.

40. The method of claim 36, wherein the spacer sequence has a length of 16 to 24 nucleotides.

41. The method of claim 36 or 40, wherein the nucleic acid molecule targeted by the guide RNA comprises a nucleotide sequence capable of complementary pairing with the spacer sequence.

42. According to the method according to any one of claims 27-41, the vector or the Cas enzyme of the system further comprises one or more nuclear localization sequences (NLS).

43. The method of any one of claims 27-42, comprising introducing the CRISPR-Cas system or the vector system into the cell via a delivery system selected from the group consisting of viral particles, liposomes, lipid nanoparticles, electroporation, microinjection, and conjugation.

44. A nucleic acid encoding the Cas enzyme of any one of claims 1-3, the fusion molecule of any one of claims 4-8, or the CRISPR-Cas system of any one of claims 9 and 11-26.

45. A cell comprising the Cas enzyme of any one of claims 1-3, the fusion molecule of any one of claims 4-8, the CRISPR-Cas system of any one of claims 9 and 11-26, the vector system of any one of claims 10-26 and / or the nucleic acid of claim 44.

46. ​​A kit comprising the Cas enzyme of any one of claims 1-3, the fusion molecule of any one of claims 4-8, the CRISPR-Cas system of any one of claims 9 and 11-26, the vector system of any one of claims 10-26, the nucleic acid of claim 44 and / or the cell of claim 33.

47. The kit of claim 46, further comprising a container for placing the Cas enzyme, the fusion molecule, the CRISPR-Cas system, the vector system, the nucleic acid and / or the cell, and instructions for use.

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