Casδ variants with improved editing efficiency and uses thereof

Abstract

The present invention relates to the field of nucleic acid editing, in particular the field of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. In particular, the present invention relates to mutants of Cas effector proteins, fusion proteins comprising said mutants, and nucleic acid molecules encoding the same. The present invention further relates to complexes and compositions for nucleic acid editing (e.g. gene or genome editing). The present invention further relates to methods for nucleic acid editing (e.g. gene or genome editing) using proteins or fusion proteins comprising the present invention. The Cas protein mutants of the present invention have better activity, e.g. higher cleavage activity, stronger target site recognition ability, higher target sequence editing activity, compared to wild type Cas proteins.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid editing, particularly to the field of regularly clustered short palindromic repeats (CRISPR) technology. Specifically, this invention relates to mutants of Cas effector proteins, fusion proteins comprising said mutants, and nucleic acid molecules encoding them. The invention also relates to complexes and compositions for nucleic acid editing (e.g., gene or genome editing). Furthermore, the invention relates to methods for nucleic acid editing (e.g., gene or genome editing) using proteins or fusion proteins comprising the invention. Background Technology

[0002] CRISPR / Cas technology, guided by RNA, can generate specific double-strand breaks at genomic target sites, and then achieve gene editing through non-homologous end joining or homologous recombination, and has become a core tool in life sciences.

[0003] Currently, CRISPR / Cas systems each have their own characteristics: Cas9 (Type II): recognizes 3′-NGG PAM, requires both crRNA and tracrRNA, and produces blunt-end breaks; Cas12a (Cpf1, Type V): recognizes 5′-TTN PAM, produces sticky ends, requires only one crRNA, and facilitates multiplex editing; CasX, CasY, and other PAM sequences are complex, limiting design flexibility; C2c1: has strict PAM restrictions (5′-TTN), target sites are easily predictable, and the potential off-target risk is low. CasX is 980 amino acids in size, while common CRISPR, C2c1, CasY, and Cpf1 are typically around 1300 amino acids in size. In short, existing CRISPR / Cas systems still have room for improvement in terms of PAM range, molecular size, RNA requirements, and multiplex editing efficiency.

[0004] Chinese patent ZL 202411237279.5 discloses a novel Cas enzyme (Casδ), and Casδ's editing of target sequences has been detected in animal cells. Therefore, optimizing the Casδ protein to further improve its editing activity and stability can enhance the application potential of Casδ. Summary of the Invention

[0005] Through extensive experimentation and repeated exploration, the inventors of this application obtained a Cas protein mutant with enhanced activity based on the wild-type Cas protein. Based on this, the inventors further developed a new CRISPR / Cas system and a gene editing method based on this system.

[0006] Cas protein mutant

[0007] Therefore, in a first aspect, this application provides a mutant of an effector protein in a CRISPR / Cas system, which contains at least one amino acid mutation compared to the effector protein shown in SEQ ID NO: 1;

[0008] The positions of the amino acid mutations contained in the mutant are selected from any one or more of the following: positions 120, 129, 413, 420, 701, 702, 710, 716, 830, 651, 672, 661, 664 and 760 corresponding to SEQ ID NO: 1.

[0009] In some embodiments, the mutant exhibits increased catalytic activity compared to the corresponding wild-type CRISPR-Cas protein (the Cas protein shown in SEQ ID NO: 1).

[0010] In some embodiments, the mutant exhibits increased gRNA binding activity compared to the corresponding wild-type CRISPR-Cas protein (the Cas protein shown in SEQ ID NO: 1).

[0011] In some embodiments, the mutant exhibits increased specificity compared to the corresponding wild-type CRISPR-Cas protein (the Cas protein shown in SEQ ID NO: 1).

[0012] In some embodiments, the mutant exhibits increased stability compared to the corresponding wild-type CRISPR-Cas protein (the Cas protein shown in SEQ ID NO: 1).

[0013] In some embodiments, the mutant exhibits reduced off-target effects compared to the corresponding wild-type CRISPR-Cas protein (the Cas protein shown in SEQ ID NO: 1).

[0014] In some embodiments, the mutant exhibits increased target-binding activity compared to the corresponding wild-type CRISPR-Cas protein (the Cas protein shown in SEQ ID NO: 1).

[0015] In some embodiments, the mutant has higher protease activity or polynucleotide binding capacity compared to the corresponding wild-type CRISPR-Cas protein (the Cas protein shown in SEQ ID NO: 1).

[0016] In some embodiments, the mutant has enhanced target sequence cleavage and / or editing activity compared to the effector protein shown in SEQ ID NO: 1.

[0017] In some embodiments, the mutant contains one amino acid mutation compared to the effector protein, and the position of the amino acid mutation in the mutant is the 120th, 129th, 420th, 702nd, 710th, 830th, 651st, 672nd or 760th position corresponding to SEQ ID NO: 1.

[0018] It is understood that this application discloses the aforementioned single mutation sites capable of enhancing the activity of wild-type Cas protein (e.g., target sequence cleavage activity and / or editing activity), and also discloses the concept of combining these single mutation sites to construct mutants containing multiple mutation sites. Based on this, those skilled in the art can, under the guidance of this application and in conjunction with existing technologies, make various different combinations of one or more mutation sites of the Cas protein and anticipate their technical effects. Therefore, this application is intended to protect Cas protein mutants containing (but not having) the aforementioned single mutation sites.

[0019] In some embodiments, the mutants described above exhibit 10%-20%, 20%-30%, 30%-40%, or more increased target sequence cleavage and / or editing activity compared to the wild-type CRISPR-Cas protein (the Cas protein shown in SEQ ID NO: 1).

[0020] In some embodiments, the mutant contains multiple amino acid mutations compared to the effector protein, and the positions of the amino acid mutations contained in the mutant correspond to positions 120 and 129 of SEQ ID NO: 1.

[0021] In some embodiments, the mutant contains multiple amino acid mutations compared to the effector protein, and the positions of the amino acid mutations contained in the mutant correspond to positions 702 and 710 of SEQ ID NO: 1.

[0022] In some embodiments, the mutant contains multiple amino acid mutations compared to the effector protein, and the positions of the amino acid mutations contained in the mutant correspond to positions 716 and 710 of SEQ ID NO: 1.

[0023] In some embodiments, the mutant contains multiple amino acid mutations compared to the effector protein, and the positions of the amino acid mutations contained in the mutant correspond to positions 702, 710, and 716 of SEQ ID NO: 1.

[0024] In some embodiments, the mutant contains multiple amino acid mutations compared to the effector protein, and the positions of the amino acid mutations contained in the mutant correspond to positions 702, 710, and 830 of SEQ ID NO: 1.

[0025] In some embodiments, the mutant contains multiple amino acid mutations compared to the effector protein, and the positions of the amino acid mutations contained in the mutant correspond to positions 710, 716, and 830 of SEQ ID NO: 1.

[0026] In some embodiments, the mutant contains multiple amino acid mutations compared to the effector protein, and the positions of the amino acid mutations contained in the mutant correspond to positions 651 and 672 of SEQ ID NO: 1.

[0027] In some implementations, mutants containing a single amino acid mutation exhibit 10%-20%, 20%-30%, 30%-40%, or more increased target sequence cleavage and / or editing activity compared to effector proteins.

[0028] In some embodiments, the mutants containing multiple mutation sites described above further enhance Cas protein activity (e.g., target sequence cleavage activity and / or editing activity) compared to mutants containing a single mutation site.

[0029] mutants HM8 and HM9

[0030] In some embodiments, the mutant contains multiple amino acid mutations compared to the effector protein, and the positions of the amino acid mutations in the mutant are positions corresponding to positions 120, 129, 420, 702, 710, 830, 651 and 672 of SEQ ID NO: 1.

[0031] Preferably, the positions of the amino acid mutations in the mutant are positions 120, 129, 420, 702, 710, 830, 651, 672 and 760 corresponding to SEQ ID NO: 1.

[0032] In some embodiments, the mutant exhibits 20%-30%, 30%-40%, 40%-50%, or more increased target sequence cleavage and / or editing activity compared to the effector protein.

[0033] In some implementations, the amino acid at the position described above is mutated to a positively charged amino acid, such as arginine or proline.

[0034] In some embodiments, the amino acid mutation at position 120 of the mutant is a D-to-F mutation. In some embodiments, the amino acid mutation at position 129 of the mutant is a K-to-F mutation. In some embodiments, the amino acid mutation at position 420 of the mutant is a K-to-R mutation. In some embodiments, the amino acid mutation at position 702 of the mutant is an S-to-R mutation. In some embodiments, the amino acid mutation at position 710 of the mutant is an A-to-R mutation. In some embodiments, the amino acid mutation at position 830 of the mutant is a V-to-R mutation. In some embodiments, the amino acid mutation at position 651 of the mutant is a K-to-R mutation. In some embodiments, the amino acid mutation at position 672 of the mutant is an L-to-R mutation. In some embodiments, the amino acid mutation at position 760 of the mutant is a P-to-R mutation. In some embodiments, the amino acid mutation at position 413 of the mutant is a K-to-R mutation. In some embodiments, the amino acid mutation at position 701 of the mutant is an A-to-R mutation. In some embodiments, the amino acid mutation at position 716 of the mutant is a G-to-R mutation. In some embodiments, the amino acid mutation at position 661 of the mutant is a K-to-R mutation. In some embodiments, the amino acid mutation at position 664 of the mutant is an E-to-R mutation.

[0035] It is understood that this application discloses the aforementioned single mutation sites that can enhance the activity of wild-type Cas proteins (e.g., target sequence cleavage activity and / or editing activity), and also discloses combined mutation sites that can enhance the activity of wild-type Cas proteins. Therefore, the focus of this application is the discovery of key mutation sites that can enhance Cas protein activity. Based on this, this application also teaches how to mutate the amino acids at the key mutation sites to positively charged amino acids, such as arginine or proline.

[0036] Therefore, those skilled in the art, guided by the teachings of this application and in conjunction with existing technologies, can make various attempts to mutate the amino acids at one or more of the aforementioned key mutation sites and anticipate their technical effects. Thus, this application is intended to protect sequences containing key mutation sites, rather than the specific sequences after mutation.

[0037] In some embodiments, the mutant contains amino acid mutations of D120F, K129R, K420R, S702R, A710R, V830R, K651R, and L672R.

[0038] In some embodiments, the amino acid sequence of the mutant is shown in SEQ ID NO: 2.

[0039] In some embodiments, the mutant contains amino acid mutations of D120F, K129R, K420R, S702R, A710R, V830R, K651R, L672R, and P760R.

[0040] In some embodiments, the amino acid sequence of the mutant is shown in SEQ ID NO: 3.

[0041] In some embodiments, the mutant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 3, and substantially retains the biological functions of the sequence from which it is derived (e.g., activity to bind to guide RNA, endonuclease activity, activity to bind to and cleave a target sequence at a specific site guided by guide RNA).

[0042] In some embodiments, the mutants containing 8 or 9 mutation sites described above, based on mutants containing one or more (e.g., 1, 2, 3, 4, 5) mutation sites, further enhance the Cas protein activity (e.g., target sequence cleavage activity and / or editing activity).

[0043] Fusion protein

[0044] The proteins of the present invention can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, protein derivatization (e.g., labeling) does not adversely affect the protein's desired activity (e.g., activity binding to guide RNA, endonuclease activity, activity of binding to and cleaving a target sequence at a specific site guided by guide RNA). Therefore, the proteins of the present invention are also intended to include such derivatized forms. For example, the proteins of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another protein or polypeptide, a detection reagent, a pharmaceutical reagent, etc.

[0045] Therefore, in a second aspect, this application provides a fusion protein comprising the mutant described in the first aspect, as well as additional proteins or polypeptides.

[0046] In some embodiments, the additional protein or polypeptide is selected from epitope tags, reporter gene sequences, nuclear localization signal (NLS) sequences, targeting moieties, transcriptional activation domains (e.g., VP64), transcriptional repression domains (e.g., KRAB or SID domains), nuclease domains (e.g., Fok1), and domains having activities selected from: nucleotide deaminase, methyltransferase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, and nucleic acid binding activity; and any combination thereof.

[0047] In some embodiments, the fusion protein of the present invention comprises one or more NLS sequences, such as the NLS of the SV40 viral large T antigen. In some embodiments, the NLS sequence is located at, near, or close to the end (e.g., N-terminus or C-terminus) of the protein or protein truncated of the present invention. In some exemplary embodiments, the NLS sequence is located at, near, or close to the C-terminus of the protein or protein truncated of the present invention.

[0048] In some embodiments, the fusion protein of the present invention includes an epitope tag.

[0049] In some embodiments, the fusion protein of the present invention comprises a reporter gene sequence.

[0050] In some embodiments, the fusion protein of the present invention includes a domain capable of binding to DNA molecules or intracellular molecules.

[0051] In some embodiments, the protein or protein truncated of the present invention is optionally fused to the additional protein or polypeptide via a linker.

[0052] In some embodiments, the additional protein or polypeptide is directly linked to the N-terminus or C-terminus of the protein or protein truncated of the present invention.

[0053] In some embodiments, the additional protein or polypeptide is connected to the N-terminus or C-terminus of the protein or protein truncated form of the present invention via a connector.

[0054] The proteins, conjugates, or fusion proteins of the present invention are not limited by their manner of production; for example, they can be produced by genetic engineering methods (recombinant technology) or by chemical synthesis methods.

[0055] Encoding nucleic acids, vectors and host cells

[0056] In a third aspect, the present invention provides an isolated nucleic acid molecule comprising: a nucleotide sequence encoding the mutant described in the first aspect and the fusion protein described in the second aspect.

[0057] In some embodiments, the nucleotide sequence is codon-optimized for expression in prokaryotic or eukaryotic cells.

[0058] In a fourth aspect, the present invention also provides a carrier comprising isolated nucleic acid molecules as described in the third aspect.

[0059] The vectors of the present invention can be cloning vectors or expression vectors. In some embodiments, the vectors of the present invention are, for example, plasmids, granules, bacteriophages, Cosmids, etc. In some embodiments, the vectors are capable of expressing the proteins, protein truncated forms, fusion proteins, isolated nucleic acid molecules as described in the fifth aspect, or complexes as described in the sixth aspect in a subject (e.g., a mammal, such as a human).

[0060] In a fifth aspect, the present invention also provides a host cell comprising the isolated nucleic acid molecule or vector as described above.

[0061] Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells (e.g., cassava, corn, sorghum, soybean, wheat, oat, or rice cells), and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). The cells of the present invention can also be cell lines, such as 293T cells.

[0062] Complex or composition

[0063] In a sixth aspect, the present invention provides a composite comprising:

[0064] (i) a protein component selected from: mutants as described in the first aspect, fusion proteins as described in the second aspect, and any combination thereof; and (ii) a nucleic acid component; wherein the protein component and the nucleic acid component are combined to form a complex.

[0065] In another aspect, the present invention provides a composition comprising:

[0066] (i) a protein component selected from: mutants as described in the first aspect, fusion proteins as described in the second aspect, and any combination thereof; and (ii) a nucleic acid component; wherein the protein component and the nucleic acid component each exist independently to form the composition.

[0067] In some implementations, the nucleic acid component is a guide RNA in a CRISPR / Cas system.

[0068] In some embodiments, the nucleic acid component comprises a unidirectional repeat sequence from the CRISPR / Cas system and a guide sequence capable of hybridizing with the target sequence from the 5' to 3' direction.

[0069] In some embodiments, the nucleic acid component comprises the sequence shown in SEQ ID NO: 4 and a guide sequence capable of hybridizing with the target sequence.

[0070] In some embodiments, the nucleic acid component comprises the sequence shown in SEQ ID NO: 4 and the sequence shown in any one of SEQ ID NO: 7 to SEQ ID NO: 21 from the 5' to 3' direction.

[0071] In some embodiments, the complex does not contain trans-acting crRNA (tracrRNA).

[0072] In some embodiments, when the target sequence is DNA, the target sequence is located at the 3' end of the protospacer adjacent motif (PAM), and the PAM has a 5'-RYR sequence, where R is selected from A or G, and Y is selected from T or C. In some embodiments, the sequence of the PAM is selected from ATG, ACG, GTG, ATA, ACA, GCA, GTA, and / or GCG.

[0073] In some implementations, when the target sequence is RNA, the target sequence does not have a PAM domain restriction.

[0074] In some embodiments, the target sequence is a DNA or RNA sequence derived from prokaryotic or eukaryotic cells. In some embodiments, the target sequence is a non-naturally occurring DNA or RNA sequence.

[0075] In some embodiments, the target sequence is present within the cell. In some embodiments, the target sequence is present in the cell nucleus or cytoplasm (e.g., organelles). In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a prokaryotic cell.

[0076] Reagent test kit

[0077] In a seventh aspect, the present invention provides a kit comprising one or more components selected from the following: mutants as described in the first aspect, fusion proteins as described in the second aspect, isolated nucleic acid molecules as described in the third aspect, vectors as described in the fourth aspect, complexes or compositions as described in the sixth aspect;

[0078] In some embodiments, the kit comprises the complex or composition described in the sixth aspect, and instructions for using the complex or composition.

[0079] In some embodiments, the components contained in the kit of the present invention can be provided in any suitable container.

[0080] In some embodiments, the kit further comprises one or more buffers. The buffer can be any buffer, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10.

[0081] Delivery and delivery composition

[0082] The mutants as described in the first aspect, the fusion proteins as described in the second aspect, the isolated nucleic acid molecules as described in the third aspect, the vectors as described in the fourth aspect, and the complexes or compositions as described in the sixth aspect of the present invention can be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipid transfection, nuclear transfection, microinjection, acoustic pore effect, gene gun, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendritic transfection, heat shock transfection, nuclear transfection, magnetic transfection, lipid transfection, puncture transfection, optical transfection, reagent-enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, viral particles, artificial viruses, etc.

[0083] Therefore, in an eighth aspect, the present invention provides a delivery composition comprising a delivery vector and one or more selected from the following: a mutant as described in the first aspect, a fusion protein as described in the second aspect, an isolated nucleic acid molecule as described in the third aspect, a vector as described in the fourth aspect, a complex or composition as described in the sixth aspect.

[0084] In some implementations, the delivery carrier is a particle.

[0085] In some embodiments, the delivery vector is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, microvesicles, gene guns, or viral vectors (e.g., replication-defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses).

[0086] method

[0087] In another aspect, the present invention provides a method for modifying a target gene, comprising: contacting the complex or composition of the sixth aspect with the target gene, or delivering it to a cell containing the target gene; wherein the target sequence is present in the target gene;

[0088] In some embodiments, the method is used to modify a target gene in vitro or ex vivo. In some embodiments, the method is not a method for treating humans or animals as a therapy. In some embodiments, the method does not include the step of modifying human germline genetic characteristics.

[0089] In some embodiments, the target gene is present within a cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is selected from non-human primate, bovine, pig, or rodent cells. In some embodiments, the cell is a non-mammalian eukaryotic cell, such as a poultry or fish cell. In some embodiments, the cell is a plant cell, such as a cell found in cultivated plants (e.g., cassava, corn, sorghum, wheat, or rice), algae, trees, or vegetables.

[0090] In some embodiments, the target gene is present in an in vitro nucleic acid molecule (e.g., a plasmid).

[0091] In some embodiments, the method results in a break in the target sequence (e.g., a double-strand break in DNA or a single-strand break in RNA). In some embodiments, the break results in a reduction in transcription of the target gene.

[0092] In some embodiments, the method further includes contacting the target gene with an editing template (e.g., a foreign nucleic acid) or delivering it to a cell containing the target gene. In such embodiments, the method repairs the broken target gene through homologous recombination with the editing template (e.g., a foreign nucleic acid), wherein the repair results in a mutation, including the insertion, deletion, or substitution of one or more nucleotides of the target gene. In some embodiments, the mutation results in a change in one or more amino acids in a protein expressed from a gene containing the target sequence.

[0093] Therefore, in some embodiments, the modification further includes inserting an editing template (e.g., exogenous nucleic acid) into the break.

[0094] In some embodiments, the protein, protein truncated form, conjugate, fusion protein, isolated nucleic acid molecule, complex, carrier, or composition is contained in a delivery vector.

[0095] In some embodiments, the delivery vector is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, and viral vectors (such as replication-defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses).

[0096] In some embodiments, the method is used to modify a cell, cell line, or organism by altering one or more target sequences in a target gene or a nucleic acid molecule encoding a target gene product.

[0097] In another aspect, the present invention provides a method for altering the expression of a gene product, comprising: contacting the complex or composition described in the sixth aspect with a nucleic acid molecule encoding the gene product, or delivering it to a cell containing the nucleic acid molecule; wherein a target sequence is present in the nucleic acid molecule.

[0098] In some embodiments, the method is used to alter the expression of a gene product in vitro or in vitro. In some embodiments, the method is not a method for treating humans or animals as a therapy. In some embodiments, the method does not include the step of modifying human germline genetic characteristics.

[0099] In some embodiments, the nucleic acid molecules are present within a cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is selected from non-human primate, bovine, pig, or rodent cells. In some embodiments, the cell is a non-mammalian eukaryotic cell, such as poultry or fish. In some embodiments, the cell is a plant cell, such as cells found in cultivated plants (e.g., cassava, corn, sorghum, wheat, or rice), algae, trees, or vegetables.

[0100] In some embodiments, the nucleic acid molecule is present in an in vitro nucleic acid molecule (e.g., a plasmid). In some embodiments, the nucleic acid molecule is present in a plasmid.

[0101] In some embodiments, the expression of the gene product is altered (e.g., enhanced or reduced). In some embodiments, the expression of the gene product is enhanced. In some embodiments, the expression of the gene product is reduced.

[0102] In some implementations, the gene product is a protein.

[0103] In some embodiments, the protein, protein truncated form, conjugate, fusion protein, isolated nucleic acid molecule, complex, carrier, or composition is contained in a delivery vector.

[0104] In some embodiments, the delivery vector is selected from lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, and viral vectors (such as replication-defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses).

[0105] In some embodiments, the method is used to modify a cell, cell line, or organism by altering one or more target sequences in a target gene or a nucleic acid molecule encoding a target gene product.

[0106] Cells and progeny

[0107] In some cases, modifications introduced into cells by the methods of the present invention can alter cells and their progeny to improve the production of their biological products (such as antibodies, starch, ethanol, or other desired cellular outputs). In other cases, modifications introduced into cells by the methods of the present invention can include changes in cells and their progeny that alter the biological products produced.

[0108] Therefore, in another aspect, the present invention also relates to cells or their progeny obtained by the method described above, wherein said cells contain modifications not present in their wild type.

[0109] In another aspect, the present invention also relates to cell products of cells or their progeny as described above.

[0110] In another aspect, the present invention also relates to an in vitro, ex vivo, or in vivo cell or cell line or its progeny, said cell or cell line or its progeny comprising: a mutant as described in the first aspect, a fusion protein as described in the second aspect, an isolated nucleic acid molecule as described in the third aspect, a vector as described in the fourth aspect, a complex or composition as described in the sixth aspect.

[0111] In some implementations, the cell is a prokaryotic cell.

[0112] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a non-human mammalian cell, such as cells of non-human primates, cattle, sheep, pigs, dogs, monkeys, rabbits, or rodents (such as rats or mice). In some embodiments, the cell is a non-mammalian eukaryotic cell, such as cells of poultry (such as chickens), fish, or crustaceans (such as clams or shrimp). In some embodiments, the cell is a plant cell, such as cells of monocotyledonous or dicotyledonous plants, or cells of cultivated plants or food crops such as cassava, corn, sorghum, soybeans, wheat, oats, or rice, such as algae, trees, or productive plants, fruits, or vegetables (e.g., trees such as citrus trees, nut trees; nightshade plants, cotton, tobacco, tomatoes, grapes, coffee, cocoa, etc.).

[0113] In some implementations, the cell is a stem cell or stem cell line.

[0114] Methods and Applications

[0115] In another aspect, the present invention relates to mutants as described in the first aspect, fusion proteins as described in the second aspect, isolated nucleic acid molecules as described in the third aspect, vectors as described in the fourth aspect, complexes or compositions as described in the sixth aspect, kits of the present invention, and their use in the preparation of formulations for: (i) nucleic acid editing (e.g., in vitro or ex vivo nucleic acid editing); (ii) in vitro or ex vivo DNA detection; and / or, (iii) editing target sequences in target loci to modify biological or non-human organisms.

[0116] In some implementations, the nucleic acid editing includes gene or genome editing.

[0117] In some implementations, the gene or genome editing includes modifying genes, knocking out genes, altering the expression of gene products, repairing mutations, and / or inserting polynucleotides.

[0118] In some embodiments, the preparation is used for the detection of single-stranded or double-stranded DNA (e.g., the detection of single-stranded or double-stranded DNA in prokaryotic cells).

[0119] In some embodiments, the DNA detection is used to detect tumors, viruses, or bacteria. Without being theoretically limited, it is believed that due to the non-specific cleavage properties of Casδ proteins or their mutants on single-stranded DNA after recognition of target DNA, when target DNA (e.g., tumor-specific markers, virus- or bacteria-specific markers) is present, detection of viruses or bacteria such as tumors, Ebola, avian influenza, and African swine fever can be achieved by adding detectable single-stranded DNA and detecting its non-specific cleavage.

[0120] In another aspect, the present invention also provides a method for detecting the presence of target nucleic acid in a sample, comprising the following steps:

[0121] (1) Contact the sample with a labeled DNA probe and any of the following components: the complex or composition as described in the sixth aspect, or the kit of the present invention;

[0122] The guide sequence contained in the complex, composition, or kit is capable of hybridizing with the target nucleic acid, and the DNA probe does not hybridize with the guide sequence; in some embodiments, the DNA probe emits a detectable signal after being cleaved.

[0123] (2) Detect a detectable signal generated by the protein or protein truncated form contained in the complex, composition or kit cleaving the DNA probe, thereby determining whether the target nucleic acid is present in the sample.

[0124] In some embodiments, one end of the DNA probe (e.g., the 5' end) is labeled with a fluorescent group, and the other end (e.g., the 3' end) is labeled with a quencher group.

[0125] In some embodiments, the target nucleic acid sequence is a sequence derived from a pathogen. In some embodiments, the pathogen is selected from viruses, bacteria, fungi, protozoa, parasites, or any combination thereof.

[0126] In some implementations, the sequence of the target nucleic acid is obtained from the genome of tumor cells.

[0127] The target nucleic acid detected in this application can be either DNA or RNA. Therefore, in some embodiments, the method further includes the step of contacting the sample with a reagent for reverse transcription. In some embodiments, the reagent for reverse transcription is selected from reverse transcriptase, oligonucleotide primers, dNTPs, or any combination thereof.

[0128] In some embodiments, the target nucleic acid is single-stranded or double-stranded. In some embodiments, the sequence of the target nucleic acid is a DNA or RNA sequence derived from prokaryotic or eukaryotic cells; or, the sequence of the target nucleic acid is a non-naturally occurring DNA or RNA sequence.

[0129] In some embodiments, the detectable signal is determined by one or more methods selected from: imaging-based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase transition / dispersion, electrochemical detection, and semiconductor-based sensing.

[0130] In some embodiments, the method further includes the step of amplifying the target nucleic acid in the sample.

[0131] Terminology Definition

[0132] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0133] In this invention, the term "Casδ" refers to a Cas effector protein that the inventors first discovered and identified, having the sequence shown in SEQ ID NO: 1. The Casδ of this invention is a nuclease that binds to and cleaves a specific site on a target sequence under the guidance of a guide RNA. In this invention, the term "Casδ mutant" refers to a Cas effector protein that the inventors first discovered and identified exhibits enhanced activity compared to Casδ.

[0134] As used herein, the terms “regularly clustered interspaced short palindromic repeats (CRISPR)-CRISPR-related (Cas) (CRISPR-Cas) system” or “CRISPR system” are used interchangeably and have the meaning commonly understood by those skilled in the art, typically comprising transcripts or other elements relating to the expression of CRISPR-related (“Cas”) genes, or transcripts or other elements capable of directing the activity of said Cas genes. Such transcripts or other elements may comprise sequences encoding Cas effector proteins and guide RNA comprising CRISPR RNA (crRNA), as well as trans-acting crRNA (tracrRNA) sequences found in the CRISPR-Cas9 system, or other sequences or transcripts derived from CRISPR loci. In the Casδ-based CRISPR system described in this invention, the tracrRNA sequence is not required.

[0135] As used herein, the terms "Cas effector protein" and "Cas effector enzyme" are used interchangeably and refer to any protein longer than 800 amino acids presented in the CRISPR-Cas system. In some cases, this category of proteins refers to proteins identified from Cas loci.

[0136] As used herein, the terms “guide RNA” and “mature crRNA” are used interchangeably and have the meanings commonly understood by those skilled in the art. Generally, a guide RNA may comprise a direct repeat sequence and a guide sequence, or consist substantially of or composed of a direct repeat sequence and a guide sequence (also referred to as a spacer sequence in the context of an endogenous CRISPR system). In some cases, the guide sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence to hybridize with said target sequence and guide the specific binding of the CRISPR / Cas complex to said target sequence. In some embodiments, the complementarity between the guide 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%, or at least 99% when optimal alignment is achieved. Determining optimal alignment is within the capabilities of those skilled in the art. For example, publicly available and commercially available alignment algorithms and programs exist, such as, but not limited to, ClustalW, the Smith-Waterman algorithm in MATLAB, Bowtie, Geneious, Biopython, and SeqMan.

[0137] In some embodiments, the guide sequence is 10-30, 15-25, 15-22, 19-25, or 19-22 nucleotides in length. In some embodiments, the direct repeat sequence is 10-40 amino acids and 15-30 nucleotides in length, for example 15-25 nucleotides, for example 20-25 nucleotides, for example 22-24 nucleotides.

[0138] As used herein, the term "CRISPR / Cas complex" refers to a ribonucleoprotein complex formed by the binding of guide RNA or mature crRNA to the Cas protein, which contains a guide sequence that hybridizes to a target sequence and binds to the Cas protein. This ribonucleoprotein complex is capable of recognizing and cleaving polynucleotides that hybridize with the guide RNA or mature crRNA.

[0139] Therefore, in the formation of a CRISPR / Cas complex, a "target sequence" refers to a polynucleotide targeted by a guide sequence designed to be targeted, such as a sequence complementary to the guide sequence, wherein hybridization between the target sequence and the guide sequence will promote the formation of a CRISPR / Cas complex. Perfect complementarity is not required, as long as sufficient complementarity exists to induce hybridization and promote the formation of a CRISPR / Cas complex. The target sequence can contain any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located in the cell nucleus or cytoplasm. In some cases, the target sequence may be located in an organelle of a eukaryotic cell, such as a mitochondrion or chloroplast. The sequence or template that can be used for recombination into a target locus containing the target sequence is called an "edit template," "edit polynucleotide," or "edit sequence." In some embodiments, the edit template is a foreign nucleic acid. In some embodiments, the recombination is homologous recombination.

[0140] In this invention, the term "target sequence" or "target polynucleotide" can refer to any endogenous or exogenous polynucleotide for a cell (e.g., a eukaryotic cell). For example, the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or useless DNA). In some cases, the target sequence is believed to be associated with a protospacer adjacent motif (PAM). The precise sequence and length requirements for the PAM vary depending on the Cas effector enzyme used, but the PAM is typically a 2-5 base pair sequence adjacent to the protospacer sequence (i.e., the target sequence). Those skilled in the art can identify the PAM sequence used with a given Cas effector protein. In this document, "specific motif sequence recognized by the Cas protein" or "motif sequence" refers to the PAM sequence.

[0141] In some cases, target sequences or target polynucleotides may include multiple disease-related genes and polynucleotides, as well as genes and polynucleotides related to signal transduction biochemical pathways. Non-limiting examples of such target sequences or target polynucleotides include those listed in U.S. Provisional Patent Applications 61 / 736,527 and 61 / 748,427, filed December 12, 2012 and January 2, 2013, respectively, and International Application PCT / US2013 / 074667, filed December 12, 2013, all of which are incorporated herein by reference.

[0142] In some cases, examples of target sequences or target polynucleotides include sequences associated with signal transduction biochemical pathways, such as signal transduction biochemical pathway-related genes or polynucleotides. Examples of target polynucleotides include disease-related genes or polynucleotides. A “disease-related” gene or polynucleotide refers to any gene or polynucleotide that produces its transcriptional or translational product at an abnormal level or in an abnormal form in cells derived from tissues affected by a disease, compared to tissues or cells from non-disease control tissues or cells. In cases where altered expression is associated with the onset and / or progression of disease, it can be a gene expressed at an abnormally high level; or it can be a gene expressed at an abnormally low level. Disease-related genes also refer to genes with one or more mutations or genetic variations that are directly responsible for or linked to one or more genes responsible for the etiology of the disease in disequilibrium. The transcribed or translated product can be known or unknown and can be at normal or abnormal levels.

[0143] As used herein, the term “wildtype” has the meaning commonly understood by those skilled in the art as referring to the typical form of an organism, strain, or gene, or the characteristic that distinguishes it from mutant or variant forms when it exists in nature, is separable from its natural source and has not been intentionally modified by humans.

[0144] As used herein, the terms “non-naturally occurring” or “engineered” are used interchangeably and indicate artificial involvement. When these terms are used to describe nucleic acid molecules or peptides, they indicate that the nucleic acid molecule or peptide is at least substantially free from at least one other component bound to it, either naturally occurring or found in nature.

[0145] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48:443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0146] As used herein, the term "hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonding between the bases of these nucleotide residues. Hydrogen bonding can occur via Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific mechanism. The complex can consist of two strands forming a duplex, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. Hybridization can constitute a step in a broader process, such as the initiation of PCR or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence is called the "complement" of that given sequence.

[0147] As used herein, the term "subject" includes, but is not limited to, various animals, such as mammals, including bovines, equines, sheep, suidae, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans. In some embodiments, the subject (e.g., a human) suffers from a condition (e.g., a condition caused by a disease-related gene defect).

[0148] Beneficial effects of the invention

[0149] Compared with existing technologies, the Cas protein mutants and systems of the present invention have significant advantages. For example, the Cas protein mutants of the present invention are smaller in size than Cas9, C2c1, CasY, and Cpf1 proteins, thus exhibiting superior transfection efficiency. For example, the PAM domain of the Cas protein mutants of the present invention has a 5'-RYR structure (R is A or G, Y is T or C), and a total of eight recognizable PAM combinations are identified: ATG, ACG, ATA, ACA, GTG, GTA, GCG, and GCA, which provides a broader range of recognition sites than the NGG PAM recognized by SpCas9 and the TTN PAM recognized by LbCas12a previously reported.

[0150] Furthermore, the Cas protein mutant of this invention exhibits better activity compared to the wild-type Cas protein, such as higher cleavage activity, stronger target site recognition ability, and higher target sequence editing activity. The target sequence editing activity is increased by an average of 3 times compared to the unoptimized version, with a maximum increase of 20.3 times.

[0151] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0152] Figure 1 The results show the editing efficiency of multiple single-point mutants.

[0153] Figure 2 The results show the editing efficiency of mutants after single-point and combined mutations of amino acid sites interacting with PAM.

[0154] Figure 3 The results show the editing efficiency of mutants after single-point and combinatorial mutations of amino acid sites that interact with RNA.

[0155] Figure 4 The results show the editing efficiency of mutants after single-point and combinatorial mutations of amino acid sites that interact with DNA.

[0156] Figure 5 Results of editing efficiency assays for mutants after single-point and combinatorial mutations of amino acid sites that interact with DNA-RNA hybrid chains.

[0157] Figure 6 Analysis of editing efficiency results after combining 8 amino acid mutations.

[0158] Figure 7 The results show the improvement in editing efficiency of Casδ-HM9 compared to Casδ-HM8.

[0159] Figure 8 Analysis of the editing efficiency of Casδ-HM8 and Casδ-HM9 at 10 targets in human cells.

[0160] Figure 9 This is a schematic diagram of the components in the expression box.

[0161] Figure 10 Analysis of the editing efficiency of Casδ-HM9 in maize protoplasts.

[0162] Sequence information

[0163] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0164] Table 1: Sequence Description

[0165] Detailed Implementation

[0166] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0167] Unless otherwise specified, the experiments and methods described in the examples are generally performed in accordance with conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in this invention can be found in Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989); *Current Protocols in Molecular Biology* (edited by FM. Ausubel et al., (1987)); and the *Methods in Enzymology* series (academic publishing company): *PCR 2: A PRACTICAL*. APPROACH (edited by MJ MacPherson, BD Hames and GR Taylor (1995)), Harlow and Lane (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (edited by R.R. Freshney (1987)).

[0168] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0169] The sources of some reagents and materials involved in the following examples are as follows:

[0170] pUC19 was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0171] The competent Escherichia coli cells TSC-E03 were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0172] DMEM basic culture medium (C11995500BT), NEAA (11140050), penicillin streptomycin (15140-122), and FBS fetal bovine serum (10091-148) were purchased from Thermo Fisher Scientific.

[0173] The LipoFiter 3.0 high-efficiency transfection reagent (HB-LF3-1000) was purchased from Hanheng Biotechnology Co., Ltd.

[0174] CTAB buffer: 16.7g CTAB powder, 68.37g NaCl, 83.5 mL 1M Tric-HCl (pH 8.0), 33.4 mL 0.5M EDTA (pH 8.0), and dH2O to a final volume of 1L.

[0175] Chloroform / isoamyl alcohol: Add 10 mL of isoamyl alcohol to 240 mL of chloroform and mix well.

[0176] Example 1. Engineered Casδ protein design and editing efficiency verification

[0177] In this embodiment, the Casδ protein (the Casδ protein in the authorized patent ZL 202411237279.5, whose amino acid series is shown in SEQ ID NO: 1) is used as the parental Cas protein to screen for mutants.

[0178] Specifically, the Casδ protein and substrate (substrate RNA sequence as shown in SEQ ID NO: 5, substrate target site sequence as shown in SEQ ID NO: 6) were co-input into AlphaFold3 for prediction. Amino acid sites that could enhance editing activity were screened, and these amino acids were mutated to positively charged amino acids (such as arginine and proline) to increase their interaction with the substrate. Further, based on the mutation sites in the prediction results, Casδ mutants containing single-point mutations were prepared, and the nucleotide sequences encoding these mutants were then constructed into protein expression vectors. Simultaneously, the expression vectors contained Puromycin resistance protein, which was transfected into HEK293T cells, and positive cells were selected using Puromycin. Subsequently, gDNA was extracted, a library was constructed, and next-generation sequencing was used to determine the editing efficiency.

[0179] To verify the editing activity of single-point mutants, the assay was performed in human HEK293T cells. Subsequently, expression vectors containing these single-point mutant genes and expression vectors containing the U6 promoter and guide RNA (containing the prototype direct repeat sequence shown in SEQ ID NO: 4 and the guide sequence in SEQ ID NO: 7) were transfected into human HEK293U cells via liposome transfection. After culturing at 37°C and 5% CO2 for 24 h, puromycin was added and the cells were cultured for another 48 h. DNA was then extracted from all cells, and the 200 bp sequence containing the target site was amplified. The PCR products were sent to Beijing Geneplus Medical Laboratory Co., Ltd. for next-generation library construction and sequencing.

[0180] The editing efficiency of wild-type Casδ protein and single-point mutants containing different amino acid mutations was detected, and the results are as follows: Figure 1 As shown. Figure 1 Different points represent the relative editing efficiency of different Cas protein single-point mutants. The results showed that the editing efficiency decreased after mutation of some amino acids, but the editing efficiency was increased by 20% or more at some amino acid sites, such as D120F, K129R, K420R, S702R, A710R, V830R, K651R, L672R, and P760R.

[0181] Example 2. Screening of combined mutants

[0182] The applicant's optimization strategy involves first performing intra-domain combinations to screen for superior combinations, and then performing inter-domain combination mutations. An expression vector containing the U6 promoter and guide RNA (containing the prototype direct repeat sequence shown in SEQ ID NO: 4 and the guide sequence contained in SEQ ID NO: 8) was then constructed using the method described in Example 1, and the editing activities of various types were again detected in HEK293T animal cells.

[0183] The amino acid sites tested in Example 1 were classified into four categories based on their functional domains: interaction with PAM, interaction with RNA, interaction with DNA, and interaction with DNA-RNA hybrid chains.

[0184] (1) Two sites, D120F and K129R, were selected to interact with PAM, and a type was formed by combining them. The results are as follows: Figure 2 As shown, the editing efficiency after the D120F+K129R combination is further improved compared with the single mutation, so the D120F+K129R combination was selected as the candidate site;

[0185] (2) Two sites, K413R and K420R, were selected to interact with RNA, and a type was created by combining them. The results are as follows: Figure 3As shown, the editing efficiency of the combination of K413R and K420R is improved compared with wild type, but not as high as that of K420R single mutant. Therefore, K420R was selected as the candidate site.

[0186] (3) Interacting with DNA, the five sites A710R, F701R, S702R, G716R, and V830R were selected, resulting in 18 possible combinations. The results are as follows: Figure 4 As shown, the top five combinations with the highest editing efficiency were selected as candidate sites: S702R+A710R, G716R+A710R, S702R+A710R+G716R, S702R+A710R+V830R, and A710R+G716R+V830R.

[0187] (4) The sites that interact with the DNA-RNA hybrid strand are selected from four sites: L672R, K651R, K661R, and E664R. Seven types of combinations are generated, and the two combinations with the highest editing efficiency are selected. Figure 5 K661R and K651R+L672R were selected as candidate sites.

[0188] All the above candidate sites were combined and mutated to form 62 different combinations. The editing activity of each combination was then tested in HEK293T animal cells, and the combination variant with the highest editing efficiency was screened. Figure 6 This variant was named Casδ-HM8 (its amino acid sequence is shown in SEQ ID NO: 2). This variant contains eight amino acid sites: D120F, K129R, K420R, S702R, A710R, V830R, K651R, and L672R.

[0189] Example 3. Optimization of Combined Mutants

[0190] To further improve editing efficiency, the applicant superimposed a P760R site on Casδ-HM8, naming it Casδ-HM9 (its amino acid sequence is shown in SEQ ID NO: 3). This variant contains nine amino acid sites: D120F, K129R, K420R, S702R, A710R, V830R, K651R, L672R, and P760R. An expression vector containing the U6 promoter and guide RNA (containing the prototype direct repeat sequence shown in SEQ ID NO: 4 and the guide sequence shown in SEQ ID NO: 7) was constructed using the method described in Example 1. Editing activities of various types were detected in HEK293T animal cells, and the results are as follows: Figure 7 As shown, Casδ-HM8 improved editing performance by 27% compared to the wild type, and Casδ-HM9 improved editing efficiency by 37% compared to the wild type.

[0191] Example 4. Detection of editing activity of Casδ mutant in human cells

[0192] In this embodiment, 10 target sites (sg1 to sg10) were selected from the genomic sequence of human HEK293T cells, and 10 guide sequences were designed for each target site (sequences shown in SEQ ID NO: 7 to SEQ ID NO: 16, respectively). Furthermore, eukaryotic expression vectors containing the Casδ-HM8 and Casδ-HM9 genes and expression vectors containing the U6 promoter and guide RNA (containing the prototype direct repeat sequence shown in SEQ ID NO: 4 and the guide sequences shown in SEQ ID NO: 7 to SEQ ID NO: 16) were transfected into human HEK293T cells using liposome transfection. After culturing at 37°C and 5% CO2 for 24 h, puromycin was added and the cells were cultured for another 48 h. DNA was extracted from all cells, and the 200 bp sequence containing the target site was amplified. The PCR products were sent to Beijing GenePlus Medical Laboratory Co., Ltd. for next-generation library construction and sequencing.

[0193] Sequencing results were statistically analyzed using bioinformatics and the original target sequences. The results showed that Casδ-HM8 and Casδ-HM9 could effectively edit 10 target sites, with an editing efficiency of up to approximately 54%. Figure 8 Compared to the wild type, the editing efficiency was improved by an average of 3 times.

[0194] Example 5. Detection of the editing activity of Casδ mutants in plants

[0195] Target design was performed on five maize target genes (where the direct repeat sequence is shown in SEQ ID NO: 4, and the guide sequences Sg11 to Sg15 are shown in SEQ ID NO: 17 to SEQ ID NO: 21). The Casδ-HM8 and Casδ-HM9 proteins were constructed into the pBUE411C vector, as shown below. Figure 9 As shown (N represents NLS, nuclear localization signal; F represents 3xFlag tag protein), crRNA expression was driven by the ZmU6 promoter, and Casδ-HM8 and Casδ-HM9 protein expression was driven by the Ubi promoter. The entire expression cassette was constructed on the PUC19 vector for transformation of protoplasts.

[0196] The extraction and transformation methods for maize protoplasts were based on the experimental methods of Yoo et al. (Yoo et al., 2007), as detailed below:

[0197] To cultivate etiolated corn seedlings, corn can be allowed to grow in the dark for 10-14 days. The middle part of the leaves with good growth is taken as the material for preparing protoplasts.

[0198] Prepare the required enzyme hydrolysate and 0.6 M mannitol equilibration solution according to the experimental needs.

[0199] After removing the leaves, stack them neatly together, cut them into thin strips with a sharp blade, and immerse them in 0.6 M mannitol for 10 minutes to equilibrate.

[0200] After equilibration, filter through a 200-mesh sieve, place the chopped leaves into the prepared enzymatic hydrolysate, and vacuum for 30 minutes.

[0201] The enzyme was digested in the dark at 28°C and 40 rpm for 3-4 hours. After digestion, it was shaken at 80 rpm for 5 minutes to separate the protoplasts as much as possible. The enzyme solution turned yellow.

[0202] Filter the enzymatically hydrolyzed solution through a 200-mesh sieve and slowly collect it into a 50 mL centrifuge tube. Centrifuge at 110 g at room temperature for 5 min with an ascending speed of 1 g and a descending speed of 1 g. Carefully remove the supernatant.

[0203] Slowly add an appropriate amount of W5 solution along the wall, gently resuspend the precipitate, centrifuge at 110 g at room temperature for 5 min, with an ascending rate of 1 g and a descending rate of 1 g, and carefully remove the supernatant.

[0204] Add an appropriate amount of W5 slowly along the wall again, gently resuspend the precipitate, and place it on ice for an ice bath for 45-60 minutes.

[0205] Use a pipette or tube to aspirate as much of the supernatant as possible, and add an appropriate amount of MMG until the protoplast count is 1-2 × 10⁻⁶. 6 / mL.

[0206] Add 60 µg of plasmid (concentration greater than 1 µg / µL) to a 2 mL centrifuge tube, then add 200 µL of protoplasts and 40% PEG (the amount of PEG is the sum of the volumes of plasmids and protoplasts), gently mix, and react at room temperature in the dark for 18 min.

[0207] After the reaction was completed, add twice the volume of W5 solution to terminate the reaction. Centrifuge at 110 g at room temperature for 5 min with an ascending rate of 1 g and a descending rate of 1 g. Carefully remove the supernatant.

[0208] Add an appropriate amount of W5 to gently resuspend the precipitate, centrifuge at 110 g at room temperature for 5 min with an ascending rate of 1 g and a descending rate of 1 g, and carefully remove the supernatant.

[0209] Add an appropriate amount of W5 solution to gently resuspend the centrifuge tube, place it flat, and incubate at 28°C in the dark for 44-48 h.

[0210] Centrifuge at 110 g at room temperature for 5 min with an ascending rate of 1 g and a descending rate of 1 g. Carefully remove the supernatant and collect the protoplasts for further experiments.

[0211] The specific preparation of the buffer solution used in the above experiment is as follows:

[0212] Preparation of the relevant stock solution (stored at 4°C):

[0213]

[0214] W5 (store at 4°C) solution:

[0215] Ingredient content

[0216]

[0217] MMG (store at 4°C) solution:

[0218]

[0219] 40% PEG (prepare fresh before use) solution:

[0220]

[0221] Preparation of enzymatic hydrolysate (15 mL):

[0222]

[0223] The above mixture was placed in a 55°C water bath for 10 min, then cooled to room temperature on ice. 15 μL of 1 M CaCl2 and 150 μL of 10% BSA were then added. After mixing, the mixture was filtered through a 0.22 μm filter for sterilization.

[0224] After culturing maize protoplasts to grow into maize plants, DNA was extracted from the maize plants, and the 200 bp sequence containing the target site was amplified. The PCR product was sent to Beijing Geneplus Medical Laboratory Co., Ltd. for next-generation library construction and sequencing. The results are as follows: Figure 10 As shown, the editing efficiency of Casδ-HM9 is 1.69-25.02%, which is 3.2-20.3 times higher than before optimization.

[0225] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A mutant of an effector protein in a CRISPR / Cas system, which is based on the effector protein shown in SEQ ID NO: 1 and undergoes any one of the following mutations: (1) D120F; (2) D120F and K129R; (3) D120F, K129R, K420R, S702R, A710R, V830R, K651R and L672R; or (4) D120F, K129R, K420R, S702R, A710R, V830R, K651R, L672R and P760R.

2. The mutant of claim 1, wherein the amino acid sequence of the mutant is shown in SEQ ID NO:

2.

3. The mutant of claim 1, wherein the amino acid sequence of the mutant is shown in SEQ ID NO:

3.

4. A fusion protein comprising the mutant according to any one of claims 1-3, and additional proteins or polypeptides; wherein, The additional protein or polypeptide is selected from epitope tags, reporter gene sequences, nuclear localization signal (NLS) sequences, and any combination thereof.

5. An isolated nucleic acid molecule comprising: a nucleotide sequence encoding a mutant of any one of claims 1-3 or a fusion protein of claim 4.

6. The isolated nucleic acid molecule of claim 5, wherein the nucleotide sequence is codon-optimized for expression in prokaryotic or eukaryotic cells.

7. A vector comprising the isolated nucleic acid molecule as described in claim 5 or 6.

8. A host cell comprising the isolated nucleic acid molecule of claim 5 or 6 or the vector of claim 7.

9. A complex or composition comprising: (i) a protein component, which is a mutant according to any one of claims 1-3; and (ii) Nucleic acid components; in, The protein component and the nucleic acid component combine to form a complex, or the protein component and the nucleic acid component exist independently to form a composition.

10. The complex or composition of claim 9, wherein the nucleic acid component comprises, from the 5' to the 3' direction, a unidirectional repeat sequence in the CRISPR / Cas system and a guide sequence capable of hybridizing with a target sequence.

11. The complex or composition of claim 9, wherein the nucleic acid component comprises, from the 5' to the 3' direction, the sequence shown in SEQ ID NO: 4 and the sequence shown in any one of SEQ ID NO: 7 to SEQ ID NO:

21.

12. The complex or composition of claim 9, wherein the complex does not contain trans-acting crRNA (tracrRNA).

13. The complex or composition of claim 10, wherein, When the target sequence is DNA, the target sequence is located at the 3' end of the adjacent motif (PAM) of the original spacer sequence, and the PAM has a sequence shown as 5'-RYR, where R is A or G and Y is T or C.

14. The complex or composition of claim 13, wherein the sequence of the PAM is selected from ATG, ACG, GTG, ATA, ACA, GCA, GTA and / or GCG.

15. The complex or composition of claim 13, wherein the target sequence is a DNA or RNA sequence derived from prokaryotic or eukaryotic cells; or, the target sequence is present in a nucleic acid molecule in vitro.

16. A kit comprising one or more components selected from the following: a mutant according to any one of claims 1-3, a fusion protein according to claim 4, an isolated nucleic acid molecule according to claim 5 or 6, a vector according to claim 7, and a complex or composition according to any one of claims 9-15.

17. A delivery composition comprising a delivery vector and one or more of the following: a mutant according to any one of claims 1-3, a fusion protein according to claim 4, an isolated nucleic acid molecule according to claim 5 or 6, a vector according to claim 7, a complex or composition according to any one of claims 9-15.

18. The delivery composition of claim 17, wherein the delivery vector is selected from lipid particles, metal particles, protein particles, exosomes, and viral vectors.

19. The delivery composition of claim 17, wherein the delivery vector is selected from replication-defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses.

20. A method for modifying a target gene for non-disease diagnostic and therapeutic purposes, comprising: The complex or composition according to any one of claims 9-15 is contacted with the target gene or delivered to a cell containing the target gene; wherein the target sequence is present in the target gene.

21. The method of claim 20, wherein the target gene is present in a cell.

22. The method of claim 20, wherein the cell is a prokaryotic cell or a eukaryotic cell.

23. The method of claim 20, wherein the cell is selected from animal cells or plant cells.

24. The method of claim 20, wherein the method causes the target sequence to break.

25. A method for altering the expression of a gene product for purposes other than disease diagnosis and treatment, comprising: The complex or composition according to any one of claims 9-15 is contacted with a nucleic acid molecule encoding the gene product, or delivered to a cell containing the nucleic acid molecule; wherein the target sequence is present in the nucleic acid molecule.

26. The method of claim 25, wherein the nucleic acid molecule is present within a cell.

27. The method of claim 25, wherein the cell is a prokaryotic cell or a eukaryotic cell.

28. The method of claim 25, wherein the cell is selected from animal cells or plant cells.

29. The method of claim 25, wherein the expression of the gene product is altered.

30. The method of claim 25, wherein the gene product is a protein.

31. Use of the mutant of any one of claims 1-3, the fusion protein of claim 4, the isolated nucleic acid molecule of claim 5 or 6, the vector of claim 7, the complex or composition of any one of claims 9-15, in the preparation of a formulation for: (i) nucleic acid editing; and / or (ii) in vitro DNA detection.

32. The use according to claim 31, wherein the nucleic acid editing includes gene editing.

33. The use according to claim 32, wherein the gene editing includes modifying a gene, knocking out a gene, altering the expression of a gene product, repairing mutations, and / or inserting polynucleotides.

34. A method for detecting the presence of a target nucleic acid in a sample for non-disease diagnosis and treatment purposes, comprising the following steps: (1) Contact the sample with a labeled DNA probe and any of the following components: The complex or composition according to any one of claims 9-15 or the kit according to claim 16; The guide sequence contained in the complex, composition, or kit is capable of hybridizing with the target nucleic acid, and the DNA probe does not hybridize with the guide sequence; wherein the DNA probe emits a detectable signal after being cleaved. (2) Detect a detectable signal generated by the mutant contained in the complex, composition or kit cleaving the DNA probe to determine whether the target nucleic acid is present in the sample.

35. The method of claim 34, wherein one end of the DNA probe is labeled with a fluorescent group and the other end is labeled with a quencher group.

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