RNA-guided nuclease and application thereof

Abstract

The invention provides a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas effector protein as well as a composition and application thereof. Wherein the CRISPR-Cas effector protein comprises a protein which has at least 80% of identity with an amino acid sequence described in any one of SEQ ID Nos.1-4; the invention also relates to complexes and compositions for nucleic acid editing comprising the proteins or fusion proteins of the invention, or nucleic acid molecules encoding them. The invention also relates to a method for nucleic acid editing using a protein or fusion protein comprising the invention.

Description

Technical Field

[0001] The present invention relates to the field of gene editing, and in particular to a Cas protein, a gene editing system and applications thereof. Background Art

[0002] The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is formed by bacteria and archaea to defend against the DNA of invading phages. The immune interference process of the CRISPR system mainly includes three stages: adaptation, expression and interference. In the adaptation stage, the CRISPR system integrates a short DNA fragment from a phage or plasmid between the leader sequence and the first repeat sequence. Each integration is accompanied by the replication of the repeat sequence, thereby forming a new repeat-spacer sequence unit. In the expression stage, the CRISPR locus is transcribed into a CRISPR RNA (crRNA) precursor (pre-crRNA), which is further processed into a small crRNA at the repeat sequence in the presence of Cas protein and tracrRNA. Mature crRNA forms a Cas / crRNA complex with Cas protein. In the interference stage, crRNA guides the Cas / crRNA complex to find the target through its complementary region to the target sequence, and causes double-stranded DNA breaks at the target site through the nuclease activity of the Cas protein, thereby making the target DNA lose its original function. CRISPR systems are divided into three families: type I, II, and type III. The most common type II system is the CRISPR / Cas9 system. The Cas9 protein can process pre-crRNA into mature crRNA that binds to tracrRNA with the assistance of trans-encoded small RNA (tracrRNA). Later, people found that by artificially constructing a single-stranded chimeric guide RNA (guideRNA) that simulates the crRNA:tracrRNA complex, the Cas9 protein can effectively mediate the recognition and cutting of the target. The three bases adjacent to the 3′ end of the target must be in the form of 5′-NGG-3′, thereby forming the PAM (protospacer and Jacent motif) structure required for the Cas / crRNA complex to recognize the target. However, the different CRISPR / Cas currently available have different advantages and disadvantages. For example, Cas9, C2c1, and CasX all require two RNAs for guide RNA. Common Cas9, C2c1, CasY, and Cpf1 are usually around 1,300 amino acids in size. In addition, the PAM sequences of Cas9, Cpf1, CasX, and CasY are complex and diverse. At present, it is still necessary to develop new Cas proteins and CRISPR-Cas systems to meet diverse application needs. Summary of the invention

[0003] The main purpose of the present invention is to provide a new Cas protein, its composition and application to meet the above application needs. Based on this, the present invention also provides a new CRISPR-Cas composition and a gene editing method and a nucleic acid detection method based on the system.

[0004] In one aspect, the present invention provides a Cas protein comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the amino acid sequences of SEQ ID Nos. 1-4, and substantially retaining the biological function of the sequence from which it is derived;

[0005] In one embodiment, the amino acid sequence of the Cas protein has one or more amino acid substitutions, deletions or additions compared to any one of the amino acid sequences of SEQ ID Nos. 1-4, and substantially retains the biological function of the sequence from which it is derived;

[0006] In one embodiment, the Cas protein comprises the amino acid sequence shown in any one of SEQ ID Nos.1-4;

[0007] or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID Nos. 1-4; or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in any one of SEQ ID Nos. 1-4;

[0008] In one embodiment, the protein has an amino acid sequence shown in any one of SEQ ID Nos. 1-4.

[0009] It is clear to those skilled in the art that the structure of a protein can be changed without adversely affecting its activity and functionality, for example, by introducing one or more conservative amino acid substitutions into the amino acid sequence of the protein without adversely affecting the activity and / or three-dimensional structure of the protein molecule.

[0010] Those skilled in the art are aware of examples and embodiments of conservative amino acid substitutions. Specifically, the amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., another non-polar amino acid residue can be substituted with a non-polar amino acid residue, another polar non-charged amino acid residue can be substituted with a polar uncharged amino acid residue, another basic amino acid residue can be substituted with a basic amino acid residue, and another acidic amino acid residue can be substituted with an acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. As long as the substitution does not lead to the inactivation of the biological activity of the protein, the conservative substitution of one amino acid being replaced by other amino acids belonging to the same group falls within the scope of the present invention. Therefore, the protein of the present invention can contain one or more conservative substitutions in the amino acid sequence, and these conservative substitutions are preferably produced by substitution according to Table 1. In addition, the present invention also covers proteins that also contain one or more other non-conservative substitutions, as long as the non-conservative substitutions do not significantly affect the desired functions and biological activities of the protein of the present invention.

[0011] Conservative amino acid substitutions can be performed at one or more predicted non-essential amino acid residues. "Non-essential" amino acid residues are amino acid residues that can be changed (deleted, substituted or replaced) without changing biological activity, while "essential" amino acid residues are required for biological activity. "Conservative amino acid substitutions" are substitutions in which amino acid residues are replaced by amino acid residues with similar side chains. Amino acid substitutions can be performed in non-conserved regions of Cas enzymes. In general, such substitutions are not performed on conserved amino acid residues, or on amino acid residues located within conserved motifs, where such residues are required for protein activity. However, it will be appreciated by those skilled in the art that functional variants may have fewer conservative or non-conservative changes in conserved regions.

[0012] In certain embodiments, selected groups of amino acids that are considered to be conservative substitutions for each other are:

[0013]

[0014] It is known to those skilled in the art that one or more amino acid residues can be changed (replaced, deleted, truncated or inserted) from the N and / or C terminus of a protein while still retaining its functional activity. Therefore, proteins that have changed one or more amino acid residues from the N and / or C terminus of the Cas protein of the present invention while retaining its desired functional activity are also within the scope of the present invention. These changes may include changes introduced by modern molecular methods such as PCR, which includes PCR amplification of a protein coding sequence by means of an amino acid coding sequence included in the oligonucleotides used in the PCR amplification to change or extend the protein coding sequence.

[0015] It will be appreciated that proteins can be altered in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions, and methods for such manipulations are generally known to those skilled in the art.

[0016] For example, amino acid sequence variants of Cas proteins can be prepared by mutation of DNA. It can also be accomplished by other forms of mutagenesis and / or by directed evolution, for example, using known mutagenesis, recombination and / or shuffling methods, combined with relevant screening methods, to perform one or more amino acid substitutions; or one to more amino acid deletions and / or one to more amino acid insertions.

[0017] Those skilled in the art will appreciate that these minor amino acid changes in the Cas proteins of the present invention can occur (e.g., naturally occurring mutations) or be produced (e.g., using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site, or other functional domain of the protein, the properties of the polypeptide may be changed, but the polypeptide may retain its activity. If the mutations present are not close to the catalytic domain, active site, or other functional domain, it can be expected that the impact will be small.

[0018] Those skilled in the art can identify the essential amino acids of the Cas protein according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis of bioinformatics systems. The catalytic domain, active site or other functional domain of the protein can also be determined by physical analysis of the structure, such as by the following techniques: such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, combined with mutations of amino acids at putative key sites.

[0019] In one aspect, the present invention provides a fusion protein comprising the Cas protein of the present invention and one or more functional domains.

[0020] In one embodiment, the functional domain includes one or more of a localization signal, a reporter protein, a Cas protein targeting portion, a DNA binding domain, an epitope tag, a transcription activation domain, a transcription repression domain, a nuclease, a deamination domain, a methylase, a demethylase, a transcription release factor, an HDAC, a cleavage active polypeptide, and a ligase;

[0021] In one embodiment, the functional domain is selected from an adenosine deaminase catalytic domain or a cytidine deaminase catalytic domain.

[0022] In one embodiment, the localization signal comprises a nuclear localization signal and / or a nuclear export signal;

[0023] Preferably, the nuclear export signal comprises human protein tyrosine kinase 2;

[0024] Preferably, the reporter protein comprises one or more of glutathione-S-transferase, horseradish peroxidase, chloramphenicol acetyltransferase, β-galactosidase, β-glucuronidase or autofluorescent protein;

[0025] Preferably, the autofluorescent protein includes one or more of green fluorescent protein, HcRed, DsRed, cyan fluorescent protein, yellow fluorescent protein or blue fluorescent protein;

[0026] Preferably, the DNA binding domain comprises one or more of methylation binding protein, LexADBD or Gal4DBD;

[0027] Preferably, the epitope tag comprises one or more of a histidine tag, a V5 tag, a FLAG tag, an influenza virus hemagglutinin tag, a Myc tag, a VSV-G tag or a thioredoxin tag;

[0028] Preferably, the transcriptional activation domain comprises VP64 and / or VPR;

[0029] Preferably, the transcriptional repression domain comprises KRAB and / or SID;

[0030] Preferably, the nuclease comprises FokI;

[0031] Preferably, the deamination domain comprises one or more of ADAR1, ADAR2, APOBEC, AID or TAD;

[0032] Preferably, the cleavage active polypeptide includes a polypeptide having single-stranded RNA cleavage activity, a polypeptide having double-stranded RNA cleavage activity, a polypeptide having single-stranded DNA cleavage activity or a polypeptide having double-stranded DNA cleavage activity; preferably, the ligase includes DNA ligase and / or RNA ligase.

[0033] In one embodiment, the functional domain is the full length or functional fragment of TadA8e;

[0034] In one aspect, the present invention provides a polynucleotide, which is a polynucleotide sequence encoding the Cas protein, or a polynucleotide sequence encoding the aforementioned fusion protein.

[0035] In one embodiment, the polynucleotide is a DNA molecule that is codon-optimized according to the codon preference of the host cell;

[0036] In one embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell;

[0037] In one embodiment, the DNA molecule comprises nucleotides having more than 70%, preferably more than 90%, more preferably more than 95%, further preferably 99%, and further preferably 100% identity with the nucleotide sequence of any one of SEQ ID Nos. 5-8.

[0038] In one aspect, the present invention provides a guide RNA, comprising a direct repeat (DR) sequence capable of binding to the Cas protein and a spacer sequence capable of targeting a target sequence.

[0039] In one embodiment, the 3' end of the direct repeat (DR) sequence comprises a stem-loop structure, and further comprises a stem of the stem-loop structure formed by hybridization of a first stem nucleotide chain and a second stem nucleotide chain, and the loop nucleotide chain forms a loop of the stem-loop structure.

[0040] In some embodiments, in some embodiments, the stem of the stem-loop structure does not need accurate base pairing. Therefore, the stem can include one or more base mispairings. Alternatively, the base pairing can be accurate, i.e., does not include any mispairing. In some embodiments, the stem structure of the stem-loop structure separates two stem parts by a ring.

[0041] In some embodiments, the direct repeat (DR) sequence does not form a stem-loop structure, that is, there is no base pairing in the entire DR sequence.

[0042] In one embodiment, the direct repeat (DR) sequence comprises a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in SEQ ID Nos. 9-12.

[0043] In one embodiment, the direct repeat (DR) sequence comprises a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID Nos. 9-12, more preferably at least 90% identical, and even more preferably at least 95% identical.

[0044] As used herein, a direct repeat sequence may refer to a DNA coding sequence in a CRISPR locus, or to an RNA encoded therein in a crRNA. Thus, when any one of SEQ ID Nos. 9-12 is mentioned in the context of an RNA molecule such as a crRNA, each T is understood to represent a U.

[0045] In one embodiment, the direct repeat sequence, the direct repeat (DR) sequence comprises a sequence shown in any one of SEQ ID Nos. 9-12.

[0046] In one embodiment, more than 80% of the spacer sequence is complementary to the target nucleic acid;

[0047] In one embodiment, more than 90%, more preferably more than 95%, further preferably more than 99%, and further preferably 100% of the spacer sequence is complementary to the target nucleic acid;

[0048] In one embodiment, the length of the spacer sequence is 18-41 nt;

[0049] In one embodiment, the spacer sequence is 20 nt in length.

[0050] In one aspect, the present invention also provides a CRISPR-Cas composition, comprising:

[0051] (1) Protein component: the aforementioned Cas protein, or the aforementioned fusion protein; or a nucleic acid molecule encoding the Cas protein or the fusion protein;

[0052] (2) RNA component: guide RNA, or one or more nucleic acids encoding the guide RNA, or a precursor RNA of the guide RNA, or a nucleic acid encoding a precursor RNA of the guide RNA;

[0053] The protein component and the nucleic acid component are combined with each other to form a complex.

[0054] In one embodiment, the composition is an activated CRISPR complex, and the activated CRISPR complex further comprises: a target sequence of a target nucleic acid bound to the guide RNA.

[0055] In one embodiment, the CRISPR-Cas composition comprises one or more vectors, wherein the one or more vectors comprise:

[0056] (1) a first regulatory element, which is operably linked to a nucleotide sequence encoding the Cas protein or a nucleotide sequence encoding the fusion protein; and

[0057] (2) a second regulatory element, wherein the second regulatory element is operably linked to a nucleotide sequence encoding the guide RNA, wherein the guide RNA comprises:

[0058] (a) a spacer sequence capable of hybridizing to a target sequence of a target nucleic acid, and

[0059] (b) a direct repeat (DR) sequence connected to the spacer sequence and capable of guiding the Cas protein to bind to the guide RNA to form a CRISPR-Cas complex targeting the target sequence;

[0060] Wherein the first regulatory element and the second regulatory element are located on the same or different vectors of the CRISPR-Cas vector system.

[0061] In one embodiment, the first regulatory element or the second regulatory element comprises a promoter, and the promoter comprises one or more of an inducible promoter, a constitutive promoter, or a tissue-specific promoter;

[0062] In one embodiment, the promoter comprises one or more of T7, SP6, T3, CMV, EF1a, SV40, PGK1, human β-actin, CAG, U6, H1, T7, T7lac, araBAD, trp, lac or Ptac;

[0063] In one embodiment, the first regulatory element and the second regulatory element are located on the same or different vectors. In one embodiment, the vector comprises a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes simplex vector or a phagemid vector;

[0064] In one embodiment, the vector comprises a plasmid vector.

[0065] In one embodiment, the target nucleic acid comprises DNA derived from a eukaryotic organism or DNA derived from a prokaryotic organism;

[0066] In one embodiment, the eukaryotic organism includes an animal or a plant;

[0067] In one embodiment, the target nucleic acid comprises non-human mammal DNA, human DNA, insect DNA, bird DNA, reptile DNA, amphibian DNA, rodent DNA, fish DNA, worm DNA, nematode DNA, or yeast DNA;

[0068] In one embodiment, the non-human mammalian DNA comprises non-human primate DNA.

[0069] In one embodiment, the present invention provides a CRISPR-Cas system comprising one or more vectors, wherein the one or more vectors comprise:

[0070] (i) a first nucleic acid, which is a nucleotide sequence encoding the Cas protein described in the present disclosure or the fusion protein described in the present disclosure; optionally, the first nucleic acid is operably linked to a first regulatory element; and

[0071] (ii) a second nucleic acid encoding a nucleotide sequence comprising a guide RNA as described herein; optionally, the second nucleic acid is operably linked to a second regulatory element;

[0072] in:

[0073] The first nucleic acid and the second nucleic acid are present on the same or different vectors;

[0074] The guide RNA is capable of forming a complex with the Cas protein or fusion protein described in (i).

[0075] In one aspect, the present invention also provides a delivery system, the delivery system comprising the Cas protein or the fusion protein, or the polynucleotide, or the CRISPR-Cas composition. In one embodiment, the delivery system further comprises a delivery vehicle, the delivery vehicle comprising nanoparticles, liposomes, exosomes, microbubbles, gene guns or electroporation devices.

[0076] In another aspect, the present invention also provides a host cell or its progeny, wherein the host cell comprises the aforementioned Cas protein, or the aforementioned fusion protein, or the aforementioned polynucleotide, or the aforementioned vector system, or the aforementioned CRISPR-Cas system, or the aforementioned composition, and the cell or its progeny comprises a modification that is not present in its wild type.

[0077] In one embodiment, the host cell comprises a non-human mammal, human, insect, bird, reptile, amphibian, rodent, fish, worm, nematode, or yeast cell.

[0078] In one aspect, the present invention also provides a multicellular organism, comprising the aforementioned cell or its progeny.

[0079] In one embodiment, the multicellular organism is an animal model or a plant model for a relevant disease.

[0080] In one aspect, the present invention provides an enzyme preparation, comprising the Cas protein described in the present disclosure, the fusion protein described in the present disclosure, the complex described in the present disclosure, the CRISPR-Cas composition described in the present disclosure, or the system described in the present disclosure, or the delivery composition described in the present disclosure.

[0081] In one aspect, the present invention also provides a method for targeting and editing a target nucleic acid, the method comprising contacting the target nucleic acid with any one of the aforementioned CRISPR-Cas systems or compositions.

[0082] In one aspect, the present invention also provides a method for non-specifically degrading single-stranded DNA after recognizing a target nucleic acid, the method comprising contacting the target nucleic acid with the aforementioned CRISPR-Cas composition.

[0083] In one aspect, the present invention also provides a method for targeting and nicking a non-spacer complementary strand of a double-stranded target nucleic acid after recognizing a spacer complementary strand of the double-stranded target nucleic acid, the method comprising contacting the double-stranded target nucleic acid with the aforementioned CRISPR-Cas system or composition.

[0084] In one aspect, the present invention also provides a method for targeting and cleaving a double-stranded target nucleic acid, the method comprising contacting the double-stranded target nucleic acid with the aforementioned CRISPR-Cas system or composition.

[0085] In one embodiment, the non-spacer sequence complementary strand of the double-stranded target nucleic acid is nicked prior to nicking the spacer complementary strand of the double-stranded DNA.

[0086] In one aspect, the present invention also provides a method for specifically editing a double-stranded nucleic acid, the method comprising contacting the following under sufficient conditions and for a sufficient amount of time,

[0087] (1) the aforementioned Cas protein or fusion protein, another enzyme having sequence-specific nicking activity, and the guide RNA, wherein the guide RNA guides the Cas protein or the fusion protein to produce a nick on the relative strand relative to the activity of the other sequence-specific nicking enzyme; and (2) the double-stranded nucleic acid; the method results in the formation of a double-strand break.

[0088] In one aspect, the invention also provides a method of editing a double-stranded nucleic acid, the method comprising contacting the following under sufficient conditions and for a sufficient amount of time:

[0089] (1) the aforementioned Cas protein, or fusion protein, and a fusion protein of a protein domain having DNA modification activity, and the RNA guide targeting the double-stranded nucleic acid; and (2) the double-stranded nucleic acid; the Cas protein of the fusion protein is modified to produce a nick on the non-target strand of the double-stranded nucleic acid.

[0090] In one embodiment, the two strands of the double stranded nucleic acid are cleaved at different sites, resulting in staggered cleavage.

[0091] In one embodiment, both strands of the double-stranded nucleic acid are cleaved at the same site, resulting in a blunt double-strand break.

[0092] In one aspect, the present invention also provides a method for targeting and cleaving a single-stranded target nucleic acid, the method comprising contacting the target nucleic acid with the CRISPR-Cas composition of any one of the preceding claims.

[0093] In one aspect, the present invention also provides a method for inducing a change in a cell state, the method comprising contacting the aforementioned CRISPR-Cas composition with the target nucleic acid in a cell.

[0094] In one embodiment, the cell state comprises apoptosis or dormancy;

[0095] In one embodiment, the cell comprises a eukaryotic cell or a prokaryotic cell;

[0096] In one embodiment, the cell comprises a mammalian cell or a plant pathogenic cell;

[0097] In one embodiment, the cell comprises a cancer cell;

[0098] In one embodiment, the cell comprises an infectious cell or a cell infected by an infectious agent;

[0099] In one embodiment, the cell comprises a virus-infected cell, a prion-infected cell; in one embodiment, the cell comprises a fungal cell, a protozoan, or a parasite cell.

[0100] In one aspect, the present invention also provides a method for detecting a target nucleic acid in a sample, the method comprising contacting the sample with the aforementioned Cas protein, guide RNA and non-target sequence; detecting a detectable signal generated by the Cas protein cutting the non-target sequence, thereby detecting the target nucleic acid; the non-target sequence does not hybridize with the guide RNA.

[0101] In one aspect, the present invention provides a kit, comprising the aforementioned Cas protein, the aforementioned fusion protein, the aforementioned polynucleotide, the aforementioned CRISPR-Cas composition, and the use of the aforementioned host cell in preparing the kit, wherein the components of the kit are in the same or different containers.

[0102] In one aspect, the present invention also provides a container, comprising the aforementioned kit.

[0103] In one embodiment, the container comprises a sterile container;

[0104] In one embodiment, the container comprises a syringe.

[0105] In one aspect, the present invention also provides the use of the aforementioned Cas protein, the aforementioned fusion protein, the aforementioned polynucleotide, the aforementioned CRISPR-Cas composition, and the aforementioned host cell in the preparation of a medicament for treating a condition or disease in a subject in need thereof.

[0106] In one embodiment, the use comprises administering the CRISPR-Cas composition to the subject or to an ex vivo cell of the subject;

[0107] In one embodiment, the spacer sequence is complementary to at least 15 nucleotides of the target nucleic acid associated with the disorder or disease, and the Cas protein or the fusion protein cleaves the target nucleic acid; in one embodiment, the disorder or disease comprises cancer or an infectious disease;

[0108] In one embodiment, the cancer comprises one or more of Wilms tumor, Ewing sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, biliary tract cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid cancer, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or urinary bladder cancer;

[0109] In one embodiment, the condition or disease comprises one or more of cystic fibrosis, pseudohypertrophic muscular dystrophy, Becker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington disease, fragile X syndrome, Friedreich's ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, hypercholesterolemia, Leber's congenital amaurosis, sickle cell disease, hypercholesterolemia, transthyretin amyloidosis, or beta thalassemia;

[0110] In one embodiment, the infectious agent of the infectious disease comprises one or more of human immunodeficiency virus, herpes simplex virus-1, or herpes simplex virus-2.

[0111] In one aspect, the present invention also provides a method for obtaining plants with target traits, comprising contacting plant cells with the aforementioned gene editing system to modify the genes of the plant cells or introduce target genes, wherein the modification or target genes can express the target traits, to obtain modified plant cells, and regenerating using the modified plant cells to obtain plants with the target traits.

[0112] In one aspect, the present invention also provides a method for identifying a target trait in a plant, wherein a target gene in a plant cell is capable of expressing the target trait, and the plant cell is contacted with the aforementioned CRISPR-Cas composition to thereby identify the target gene.

[0113] In one aspect, the present invention also provides an implantable device, comprising the CRISPR-Cas composition.

[0114] In one embodiment, the CRISPR-Cas system or composition is within a matrix;

[0115] In one embodiment, the CRISPR-Cas system or composition is within a reservoir.

[0116] Beneficial Effects of the Invention

[0117] Compared with the Cas enzymes disclosed in the prior art, the Cas protein of the present invention is a brand-new Cas enzyme, which exhibits good nuclease activity in vivo and in vitro and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 A map of the T7-Cas-T7-crRNA vector is shown.

[0119] Figure 2 The secondary structure predictions of the direct repeat (DR) sequences corresponding to YTGE-154, YTGE-161, YTGE-164 and YTGE-165 are shown.

[0120] Figure 3 The Target plasmid map is shown.

[0121] FIG. 4 shows the editing efficiency of YTGE-154, YTGE-161, YTGE-164, and YTGE-165 in E. coli.

[0122] Figure 5 The cleavage efficiency of YTGE-154, YTGE-161, YTGE-164 and YTGE-165 in mammalian cells is shown. Specific implementation plan

[0123] The following examples are only used to describe the present invention, but not to limit the present invention. Unless otherwise specified, the experiments and methods described in the examples are basically carried out according to conventional methods well known in the art and described in various references.

[0124] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0125] Cas proteins

[0126] In the present invention, Cas protein, Cas enzyme, and Cas effector protein can be used interchangeably. Cas protein is taken in the broadest sense, including wild-type Cas protein, its derivatives or variants, analogs, and functional fragments thereof such as oligonucleotide binding fragments.

[0127] The term "wild type" has the meaning generally understood by those skilled in the art, which refers to the typical form of an organism, strain, gene, protein, or the characteristics that distinguish it from mutant or variant forms when it exists in nature, which can be isolated from a source in nature and has not been intentionally modified by man.

[0128] In some embodiments, the Cas proteins of the present invention are called YTGE-154, YTGE-161, YTGE-164 and YTGE-165, and their amino acid sequences are shown in SEQ ID Nos. 1-4.

[0129] The terms "variant", "derivative" and "analog" refer to polypeptides that substantially retain the function or activity of the Cas protein of the present invention.

[0130] In some embodiments, the Cas protein comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to the amino acid sequence shown in any one of SEQ ID Nos. 1-4.

[0131] In some embodiments, the amino acid sequence of the Cas protein has a sequence of one or more amino acid substitutions, deletions or additions compared to any one of the amino acid sequences of SEQ ID Nos. 1-4, and substantially retains the biological function of the sequence from which it is derived; the one or more amino acid substitutions, deletions or additions include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions, deletions or additions.

[0132] In some embodiments, the Cas protein and YTGE-154, YTGE-161, YTGE-164 and YTGE-165 are derived from the same species or different species.

[0133] Typically, the derivatization of a protein does not adversely affect the desired activity of the protein (e.g., activity binding to a guide RNA, endonuclease activity, activity binding to and cutting a specific site of a target sequence under the guidance of a guide RNA), that is, the derivative of the protein has the same activity as the protein. A modified form of a "derivative" includes one or more amino acids of the protein that may be deleted, inserted, modified, and / or substituted. The terms "non-naturally occurring" or "engineered" are used interchangeably and indicate artificial involvement.

[0134] Orthologue (ortholog)

[0135] As used herein, the term "orthologue" has the meaning commonly understood by those skilled in the art. As a further guide, an "orthologue" of a protein as described herein refers to a protein belonging to a different species that performs the same or similar function as its orthologue.

[0136] The nucleic acid cleavage of the present invention includes: DNA or RNA breakage in the target nucleic acid produced by the Cas protein (Cis cleavage), DNA or RNA breakage in the side branch nucleic acid substrate (single-stranded nucleic acid substrate) caused by the Cas protein side cutting activity (i.e. non-specific or non-targeted, Trans cleavage). In some embodiments, the cleavage is a double-stranded DNA break. In some embodiments, the cleavage is a single-stranded DNA break or a single-stranded RNA break.

[0137] Trans cutting refers to that in certain environments, the partially activated Cas protein remains active after binding to the target sequence, and continues to non-specifically cut non-target oligonucleotides. The side cutting activity can detect the presence of specific target oligonucleotides using the Cas system. For example, the Cas12i system is engineered to non-specifically cut ssDNA or transcripts. Side cutting activity is used for a highly sensitive and specific nucleic acid detection platform called SHERLOCK, which can be used for many clinical diagnoses (Gootenberg, JS et al., Nucleic acid detection with CRISPR-Cas13a / C2c2. Science 356, 438-442 (2017)).

[0138] CRISPR system

[0139] The terms “Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system” or “CRISPR system” are used interchangeably and have the meaning commonly understood by those skilled in the art, which generally includes transcription products or other elements related to the expression of CRISPR-associated (“Cas”) genes, or transcription products or other elements capable of directing the activity of the Cas genes.

[0140] CRISPR / Cas complex

[0141] The term "CRISPR / Cas complex" refers to a complex formed by the binding of gRNA (guide RNA) or mature crRNA (or guide RNA) to the Cas protein, which includes a spacer sequence hybridized to the target sequence and a co-directional repeat sequence bound to the Cas protein, and the complex is capable of recognizing and cleaving target nucleotides that can hybridize with the guide RNA or mature crRNA.

[0142] Guide RNA (gRNA, guideRNA)

[0143] The terms "guide RNA (gRNA)", "mature crRNA", "guide sequence", "guide RNA" are used interchangeably and have meanings commonly understood by those skilled in the art. In general, the guide RNA may comprise a direct repeat (DR) sequence and a spacer sequence, or may consist essentially of or consist of a direct repeat (DR) sequence and a spacer sequence.

[0144] In some cases, the 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 complex to the target sequence. In one embodiment, when optimally aligned, the degree of 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%. .

[0145] The gRNAs of YTGE-154, YTGE-161, YTGE-164 and YTGE-165 of the present invention comprise a spacer sequence that hybridizes with a target nucleic acid, wherein the target nucleic acid comprises a sequence located at the 3' end of a protospacer adjacent motif (PAM);

[0146] In some embodiments, the PAM sequence is 5'-TTN-3', wherein N is selected from A, T, C or G.

[0147] Target nucleic acid

[0148] In the present invention, target nucleic acid is used interchangeably with target sequence or target nucleic acid sequence, and refers to a specific nucleic acid comprising a nucleic acid sequence that is fully or partially complementary to the spacer sequence in the guide RNA. "Target sequence" refers to a polynucleotide targeted by the spacer sequence in the guide RNA, such as a sequence complementary to the spacer sequence, wherein the hybridization between the target sequence and the spacer sequence will promote the formation of a CRISPR-Cas complex (including Cas protein and guide RNA). Complete complementarity is not required, as long as there is enough complementarity to cause hybridization and promote the formation of a CRISPR-Cas complex. In some embodiments, the target nucleic acid comprises a non-coding region (e.g., a promoter or terminator). In some embodiments, the target nucleic acid is single-stranded, or double-stranded.

[0149] The target sequence can comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located in a cell or outside the cell. In some cases, the target sequence is located in the nucleus, cytoplasm, or organelle (e.g., mitochondria or chloroplast) of the cell.

[0150] The target nucleic acid can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). In some cases, the target sequence should be associated with a protospacer adjacent motif (PAM).

[0151] Donor template

[0152] In the present invention, donor template nucleic acid or donor template are used interchangeably to refer to a nucleic acid molecule that one or more cellular proteins can use to change the structure of the target nucleic acid after the Cas protein described herein changes the target nucleic acid.

[0153] In some embodiments, the donor template nucleic acid is a double-stranded nucleic acid or a single-stranded nucleic acid. In some embodiments, the donor template nucleic acid is linear or circular (e.g., plasmid). In some instances, the donor template nucleic acid is an exogenous nucleic acid molecule. In some instances, the donor template nucleic acid is an endogenous nucleic acid molecule (e.g., chromosome). In some embodiments, the donor template can be used to realize genetic recombination, and the recombination is homologous recombination.

[0154] Cutting

[0155] Cleavage refers to DNA breaks in a target nucleic acid produced by a Cas protein as described herein. In some embodiments, cleavage is a double-stranded DNA break. In some embodiments, cleavage is a single-stranded DNA break.

[0156] In the present invention, the meanings of cleaving a target nucleic acid or modifying a target nucleic acid may overlap. Modifying a target nucleic acid includes not only modification of a single nucleotide, but also insertion or deletion of a nucleic acid fragment.

[0157] Reporter nucleic acid

[0158] Reporter nucleic acid refers to a molecule that can be cut or otherwise deactivated by an activated CRISPR system protein as described herein. Reporter nucleic acid comprises a nucleic acid element that can be cut by a CRISPR protein (e.g., using a single-stranded non-targeted nucleic acid molecule, with different reporter groups or labeling molecules at both ends). The cutting of the nucleic acid element produces a detectable signal. Before cutting, or when the reporter nucleic acid is in an "active" state, the reporter nucleic acid prevents the generation or detection of a positive detectable signal. It will be understood that in certain example embodiments, a minimum background signal can be generated in the presence of an active reporter nucleic acid. A positive detectable signal can be any signal that can be detected using optics, fluorescence, chemiluminescence, electrochemistry, or other detection methods known in the art. For example, in certain embodiments, when a reporter nucleic acid is present, a first signal (i.e., a negative detectable signal) can be detected, which is then converted to a second signal (e.g., a positive detectable signal) after the target molecule is detected and cut or deactivated by an activated CRISPR protein. The reporter nucleic acid can be a single-stranded DNA molecule, a single-stranded RNA molecule, or a single-stranded DNA-RNA hybrid.

[0159] The detection method of the present invention can be used for quantitative detection of target nucleic acid to be detected. The quantitative detection index can be quantified according to the signal strength of the reporter group, such as according to the luminescence intensity of the fluorescent group, or according to the width of the color band.

[0160] Functional domain

[0161] In the present invention, the functional domain takes its broadest meaning, including proteins such as enzymes or factors themselves or having specific functional fragments / domains. The Cas protein (e.g., dCas protein) is connected / concluded with one or more functional domains, and the functional domains are selected from one or more of the localization signal, reporter protein, Cas protein targeting part, DNA binding domain, epitope tag, transcription activation domain, transcription inhibition domain, nuclease, deamination domain, methylase, demethylase, transcription release factor, HDAC, cleavage active polypeptide, ligase. When more than one functional domain is included, the functional domains may be the same or different.

[0162] Deamination domain

[0163] In the present invention, the deamination domain includes a deaminase (e.g., an adenosine deaminase or a cytidine deaminase) catalytic domain. As used herein, "adenosine deaminase" or "adenosine deaminase protein" refers to a protein, a polypeptide, or one or more functional domains of a protein or polypeptide that is capable of catalyzing a hydrolytic deamination reaction that converts adenine (or the adenine portion of a molecule) into hypoxanthine (or the hypoxanthine portion of a molecule), as shown below. In some embodiments, the adenine-containing molecule is adenosine (A), and the hypoxanthine-containing molecule is inosine (I). The adenine-containing molecule can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0164] Adenosine deaminases include, but are not limited to, members of the enzyme family known as adenosine deaminases acting on RNA (ADAR), members of the enzyme family known as adenosine deaminases acting on tRNA (ADAT), and other family members containing adenosine deaminase domains (ADAD). According to the present disclosure, adenosine deaminases are capable of targeting adenine in RNA / DNA and RNA duplexes. In specific embodiments, adenosine deaminases have been modified to increase their ability to edit DNA in RNA / DNA heteroduplexes of RNA duplexes.

[0165] In some embodiments, the deaminase is a cytidine deaminase. The term "cytidine deaminase" or "cytidine deaminase protein" refers to a protein, a polypeptide, or one or more functional domains of a protein or polypeptide that is capable of catalyzing a hydrolytic deamination reaction that converts cytosine (or the cytosine portion of a molecule) into uracil (or the uracil portion of a molecule). In some embodiments, the cytosine-containing molecule is cytidine (C), and the uracil-containing molecule is uridine (U). The cytosine-containing molecule can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0166] Cytidine deaminases include, but are not limited to, members of the enzyme family known as apolipoprotein B mRNA editing complex (APOBEC) family deaminases, activation-induced deaminases (AID), or cytidine deaminase 1 (CDA1). In specific embodiments, APOBEC family deaminases.

[0167] Identity

[0168] "Identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. "Identity" means the percentage of the number of identical residues between the polypeptide or nucleic acid sequences to the total number of residues, and the calculation of the total number of residues is determined based on the type of 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.

[0169] Taking a polypeptide sequence as an example, if the mutation type is one or more of the following: substitution / replacement of one or more amino acids / nucleotides, insertion within the sequence, and deletion within the sequence, the total number of residues is calculated as the larger of the molecules being compared. If the mutation type also includes an insertion (extension) at either or both ends of the sequence or a deletion (truncation) at either or both ends of the sequence, the number of amino acids inserted or deleted at either or both ends (for example, the number of insertions or deletions at both ends is less than 20) is not included in the total number of residues. When calculating the percent identity, the sequences being compared are 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. The same applies to the calculation of nucleotide identity.

[0170] Carrier

[0171] A vector is a nucleic acid molecule that is capable of transporting another nucleic acid molecule to which it has been linked.

[0172] Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, or no free ends (e.g., circular); nucleic acid molecules including DNA, RNA, or both; and other various polynucleotides known in the art. Vectors can be introduced into host cells by transformation, transduction, or transfection so that the genetic material elements they carry are expressed in the host cells. A vector can be introduced into a host cell to produce transcripts, proteins, or peptides, including proteins, fusion proteins, isolated nucleic acid molecules, etc. as described herein (e.g., CRISPR transcripts, such as nucleic acid transcripts, proteins, or enzymes). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. The vector may also contain a replication initiation site.

[0173] Vectors include plasmids and viral vectors, wherein the plasmid refers to a circular double-stranded DNA loop into which other DNA fragments can be inserted, for example, by standard molecular cloning techniques. Viral vectors, wherein virally derived DNA or RNA sequences are present in vectors for packaging viruses, and viruses include, for example, retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses. Viral vectors also include polynucleotides carried by viruses for transfection into a host cell. Some vectors (for example, bacterial vectors and episomal mammalian vectors with bacterial replication origins) can replicate autonomously in the host cells into which they are introduced.

[0174] Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, and are replicated together with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to as "expression vectors."

[0175] 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 level of expression desired, and the like.

[0176] Regulatory elements

[0177] "Regulatory elements" include promoters, enhancers, internal ribosome entry sites (IRES) and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals, poly-U sequences), which are described in detail in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, California (1990). In some cases, regulatory elements 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 expression of the nucleotide sequence only in certain host cells. Tissue-specific promoters can mainly direct expression in the desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas) or special cell types (e.g., lymphocytes). In other cases, regulatory elements can also direct expression in a timing-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.

[0178] "Promoter" refers to a non-coding nucleotide sequence located upstream of a gene that can initiate downstream gene expression. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or limiting a gene product, will result in the production of a gene product in the cell under most or all physiological conditions of the cell. An inducible promoter refers to a promoter that selectively expresses a coding sequence or functional RNA in response to the presence of endogenous or exogenous stimuli, such as by responding to chemical compounds (chemical inducers), or to environmental, hormone, chemical, and / or developmental signals. Inducible or regulated promoters include promoters that are induced or regulated, for example, by light, heat, stress, flooding or drought, salt stress, osmotic stress, plant hormones, wounds, or chemicals (such as ethanol, abscisic acid (ABA), jasmonate, salicylic acid, or safeners).

[0179] Host cells

[0180] "Host cell" refers to a eukaryotic cell (e.g., animal cell, plant cell, fungal cell, etc.), a prokaryotic cell (e.g., some microbial cells, E. coli, Bacillus subtilis, etc.), or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which serves as a recipient of nucleic acid (e.g., an expression vector), and includes descendants of the original cell that has been genetically modified by the nucleic acid. It should be understood that the descendants of a single cell may be due to natural, accidental, or deliberate mutations and do not necessarily have exactly the same morphology or genome, etc. as the original parent cell. "Recombinant host cells" (also called "genetically modified host cells") are host cells into which heterologous nucleic acids, such as expression vectors, have been introduced.

[0181] In some embodiments, the host cell is from a plant or animal.

[0182] In some embodiments, the plant is a dicot. In some embodiments, the dicot is selected from soybean, cabbage (cabbage) (e.g., Chinese cabbage), rapeseed, Brassica, watermelon, melon, potato, tomato, tobacco, eggplant, pepper, cucumber, cotton, alfalfa, eggplant, grape.

[0183] In some embodiments, the plant is a monocot, illustratively, the monocot is selected from rice, corn, wheat, barley, oats, sorghum, millet, grasses, Poaceae, Zizania, Avena, Coix, Hordeum, Oryza, Panicum (e.g., Panicum miliaceum), Secale, Setaria (e.g., Setaria italica), Sorghum, Triticum, Zea, Cymbopogon, Saccharum (e.g., Saccharum officinarum), Phyllostachys, Dendrocalamus, Bambusa, and Yushania.

[0184] In some embodiments, the animal is selected from pigs, cattle (ox), sheep, goats, mice, rats, alpacas, monkeys, rabbits, chickens, ducks, geese, fish (e.g., zebrafish). In some embodiments, the cell is a eukaryotic cell, injected into a mammalian cell, including a human cell (primary human cell or an established human cell line). In some embodiments, the cell is a non-human mammalian cell, such as a cell from a non-human primate (e.g., monkey), cow / bull / cow, sheep, goat, pig, horse, dog, cat, rodent (such as rabbit, mouse, rat, hamster, etc.). In some embodiments, the cell is from fish (such as salmon), birds (such as poultry, including chickens, ducks, geese), reptiles, shellfish (e.g., oysters, clams, lobsters, prawns), insects, worms, yeast, etc. In some embodiments, the cell is from a plant, such as a monocot or dicot. In certain embodiments, plant is a food crop, such as barley, cassava, cotton, groundnut or peanut, maize, millet, oil palm fruit, potato, dry bean, rapeseed or canola (canola), rice, rye, sorghum, soybean, sugar cane, beet, sunflower and wheat. In certain embodiments, plant is cereal (barley, maize, millet, rice, rye, sorghum and wheat). In certain embodiments, plant is tuber (cassava and potato). In certain embodiments, plant is sugar crop (beet and sugar cane). In certain embodiments, plant is oil-containing crop (soybean, groundnut or peanut, rapeseed or canola, sunflower and oil palm fruit). In certain embodiments, plant is fiber crop (cotton). In certain embodiments, plant is trees (such as peach or nectarine tree, apple or pear tree, nut tree (such as almond tree or walnut tree or pistachio tree) or citrus tree (for example, orange tree, grapefruit tree or lemon tree)), grass, vegetable, fruit or algae. In certain embodiments, the plant is a Solanum; a Brassica; a Lactuca; a Spinacia; a Capsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.

[0185] NLS

[0186] NLS refers to "nuclear localization sequence" or "nuclear localization signal", which refers to an amino acid sequence that promotes protein entry into the nucleus. Nuclear localization sequences are known in the art (e.g., Plank et al. International PCT Application PCT / EP2000 / 011690 filed on November 23, 2000 and published as WO / 2001 / 038547 on May 31, 2001) and are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, such as described in Koblan et al., Nature Biotech.2018doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence: KRTADGSEFESPKKKRKV, AVKRPAATKKAGQAKKKKLD, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or

[0187] MDSLLMNRKFLYQFKNVRWAKGRRETYLC.

[0188] Operatively connected

[0189] "Operably linked" means that the target nucleotide sequence is linked to the regulatory elements in a manner that allows the nucleotide sequence to be expressed (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). Advantageous vectors include lentiviruses and adeno-associated viruses, and the type of these vectors can also be selected to target specific types of cells.

[0190] Complementarity

[0191] "Complementarity" refers to the ability of one nucleic acid sequence to form one or more hydrogen bonds with another nucleic acid sequence by means of traditional Watson-Crick or other non-traditional types. The percentage of complementarity represents the percentage of residues in one nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with another nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are complementary, then the percentage of complementarity is 50%, 60%, 70%, 80%, 90% and 100%). "Complete complementarity" means that all consecutive residues of one nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in another nucleic acid sequence. "Substantially complementary" refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions.

[0192] The term "stringent conditions" in relation to hybridization refers to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes to the target sequence and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and depend on many factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.

[0193] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding of the bases between the nucleotide residues. The complex may comprise two strands forming a duplex, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a broader process such as the initiation of PCR, or cleavage of a polynucleotide by an enzyme. Sequences that are capable of hybridizing to a given sequence are referred to as the "complement" of the given sequence.

[0194] Hybridization of the target sequence with the gRNA means that at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the nucleic acid sequences of the target sequence and the gRNA can hybridize to form a complex; or represents that at least 12, 15, 16, 17, 18, 19, 20 or more bases of the nucleic acid sequences of the target sequence and the gRNA can complementarily pair and hybridize to form a complex.

[0195] Express

[0196] Nucleic acid expression includes one or more of generation of an RNA template from a DNA sequence (e.g., transcription), processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing), translation of the RNA into a polypeptide or protein, or post-translational modification of the polypeptide or protein.

[0197] deliver

[0198] "Delivery" refers to providing an entity (such as a drug) to a destination, for example, the components of the CRISPR-Cas system / composition of the present invention can be delivered in various forms, such as a combination of DNA / RNA or RNA / RNA or protein RNA. For example, the Cas protein can be delivered as a polynucleotide encoding DNA or a polynucleotide encoding RNA or as a protein.

[0199] Connectors

[0200] "Linker" refers to a linear polypeptide formed by connecting multiple amino acid residues through peptide bonds. The linker can be an artificially synthesized amino acid sequence or a naturally occurring polypeptide sequence.

[0201] treat

[0202] "Treatment" refers to treating or curing a subject's condition, delaying the onset of symptoms of a condition, and / or delaying the severity of a condition. The term "subject" includes, but is not limited to, various animals, plants, and microorganisms. Animals include mammals, such as bovines, equines, ovines, swine, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans. In certain embodiments, the subject (e.g., human) suffers from a condition (e.g., a condition caused by a disease-related genetic defect). "Plant" is any differentiated multicellular organism capable of photosynthesis, including crop plants at any maturity or developmental stage.

[0203] Example 1. Acquisition of Cas protein

[0204] The inventors analyzed the uncultured metagenome and identified 6 new Cas proteins through de-redundancy, protein clustering and other analyses. Blast analysis results showed that the sequence similarity of the Cas proteins to the reported Cas proteins was low, and these Cas proteins were named YTGE-154, YTGE-161, YTGE-164 and YTGE-165 in the present invention.

[0205] The amino acid sequences of the above-mentioned Cas proteins are shown in SEQ ID Nos. 1-4, and the nucleic acid sequences after human codon optimization are shown in SEQ ID NOs. 5-8. The direct repeat (DR) sequences of the guide RNAs corresponding to the above-mentioned proteins were analyzed, and the DR sequences of the various Cas proteins were determined to be shown in SEQ ID Nos. 9-12. The inventors used RNAfold to further analyze the RNA secondary structures of the various DR sequences. The analysis results are shown in Figure 2 Through the PAM library depletion experiment (the experiment was performed according to any embodiment of Karvelis et al. Methods. 2017 May 15; 121-122: 3-8 (the entire content of which is incorporated herein by reference), it was identified that the PAM corresponding to each of the above Cas proteins was 5'-TTN-3'.

[0206] Example 2. Verification of Cas protein cleavage activity

[0207] 1. Plasmid construction

[0208] (1) Taking the TTR gene target as the targeting object, designing the spacer sequence TTR-spacer (SEQ ID NO.13), and designing the TTR-crRNA sequence of each Cas protein, as shown in Table 1 (the crRNA sequence structure is 5'-DR sequence-spacer sequence-3').

[0209] Table 1

[0210]

[0211] The coding nucleotides of YTGE-154, YTGE-161, YTGE-164 and YTGE-165 (SEQ ID Nos. 5-8) and the corresponding TTR-crRNA were cloned into pcDNA3.1-HA (Addgene, #128034), and the crRNA was regulated by T7 promoter to obtain the recombinant expression plasmid: T7-Cas-T7-crRNA vector (see vector map for details). Figure 1 ).

[0212] (2) Suzhou Hongxun Biotechnology Co., Ltd. synthesized the araC-pBAD-CCDB fragment (SEQ ID NO.18) with the TTR target sequence (SEQ ID NO.13), and inserted the araC-pBAD-CCDB fragment into the 1284-1300 site of the pKESK22 (Addgene, Plasmid #64857) plasmid to obtain the Target plasmid. The sequence of the Target plasmid is shown in SEQ ID NO.19, and the plasmid map is shown in Figure 3 .

[0213] 2. Preparation and transformation of E. coli competent cells

[0214] The Target plasmid was transferred into DH5a competent cells and inoculated with an inoculation loop to separate and inoculate in 50

[0215] μg / ml kanamycin sulfate LB solid medium. Place in a biochemical incubator at 37°C overnight culture, pick a single colony from the plate the next day, inoculate into a LB liquid medium test tube containing 4ml 50μg / ml kanamycin sulfate (Sangon Biotechnology, A100408-0100), shake culture overnight at 37°C, 200rpm. The next day, take 4ml of the bacterial solution and inoculate into a 2L shake flask containing 400ml 50μg / ml kanamycin sulfate LB liquid medium, and culture at 37°C, 200rpm shaking for 2-3 hours.

[0216] When the OD600nm value of the bacterial solution reaches 0.3-0.5, take out the shaking bottle and place it on ice for 10-15 minutes. Pour the bacterial solution into a pre-cooled 500ml centrifuge bottle under sterile conditions, centrifuge at 4℃ and 3000rpm for 8 minutes, discard the supernatant, add about 200ml of pre-cooled CaCl2 solution, mix by blowing to suspend the bacteria, and place it in an ice bath for 30 minutes. Then centrifuge the bacterial solution at 4℃ and 3000rpm for 8 minutes, discard the supernatant, add about 8ml of pre-cooled CaCl2 solution, resuspend the bacteria, and divide the resuspended bacteria into 1.5ml EP tubes, 110μl per tube, and store them in an ultra-low temperature refrigerator at -80℃ for use.

[0217] 3. Determination of editing efficiency in E. coli

[0218] 100 ng of T7-Cas-T7-crRNA vector was transferred into Escherichia coli competent cells. After half an hour of recovery, the cells were plated on plates (CL) supplemented with carbenicillin disodium (Sangon Biotech, A100358-0001) and L-arabinose (Sangon Biotech, A610071-0100) and plates (CK) supplemented with kanamycin sulfate (Sangon Biotech, A100408-0100) and carbenicillin disodium (Sangon Biotech, A100358-0001), and cultured overnight for 16 hours. If the Cas protein cuts the target plasmid, the bacteria survive. The number of clones on the CL plate / the number of clones on the CK plate is the editing efficiency. The editing efficiency of each Cas protein was tested in the above manner, and the results obtained are as follows: Figures 4A-4B As shown in the figure, the analysis showed that each Cas protein, such as YTGE-154, YTGE-161, YTGE-164, and YTGE-165, had significant cleavage activity.

[0219] Example 3. Targeting of individual nucleases to mammalian cells

[0220] To evaluate the cleavage activity of each nuclease in mammalian cells, the applicant cloned the encoding nucleotides of each nuclease such as YTGE-154, YTGE-161, YTGE-164, and YTGE-165 (SEQ ID NO. 5-8) into the pcDNA3.1 (Invitrogen, V79020) backbone to obtain the nuclease expression plasmid. The HAO1 gene and the TTR gene were selected as targets, and the target sequences are shown in the following table:

[0221] Table 2

[0222] Target Gene Target sequence HAO1 ATTCTAGATGGAAGCTGTAT(SEQ ID NO.20) TTR TAGAAGGGATATACAAAGTG(SEQ ID NO.21)

[0223] Design the corresponding crRNA sequences as shown in the following table:

[0224] Table 3

[0225]

[0226]

[0227] The crRNA was cloned into the pUC19 vector (NEB, N3041L) and regulated by the U6 promoter to obtain the crRNA expression plasmid. The constructed plasmids were then transfected into Escherichia coli DH5a competent cells (Weidi Biotechnology, DL1001). After a large number of amplifications, the plasmids were extracted to determine the concentration and stored in a -20°C refrigerator for later use. HEK293T cells (purchased from ATCC) were inoculated in DMEM medium (Gibco, 11965092) supplemented with 10% FBS (v / v), containing 1% Penicillin Streptomycin (v / v) (Gibco, 15140122), and cultured in a 37°C cell culture incubator containing 5% CO2. The cells used for transfection were inoculated in a 24-well cell culture plate for culture the day before, and the cells were observed the next day. When the cells grew to a cell density of about 80%, transfection was performed.

[0228] The nuclease expression plasmid, the corresponding crRNA expression plasmid and the EGFP-C1 (Addgene, Plasmid, #54759) plasmid were transfected into HEK293T cells. The amount of plasmids transfected per well of the 24-well plate was 0.3 μg of nuclease expression plasmid, 0.3 μg of crRNA expression plasmid, and 0.3 ug of EGFP-C1 plasmid. The specific transfection operation is as follows:

[0229] The nuclease expression plasmid, crRNA expression plasmid, and EGFP-C1 plasmid were mixed separately and then washed with 25 μl of Dilute the transfection-specific reduced serum medium (Source Bio, L530KJ), add 2 μl of Lipofectamine 3000 (Invitrogen, L3000015) reagent, mix well by pipetting as reagent A, and let stand for 5 minutes. Dilute and mix the reduced serum medium for transfection (Source Biotechnology, L530KJ) as reagent B and let stand for 5 minutes.

[0230] Mix the above reagents A and B and blow evenly, and let it stand for 20 minutes. After the standing period, add the mixed reagent dropwise to the 24-well plate cells to be transfected and return to 37°C, 5% CO 2 The cells were cultured in an incubator. Six hours after transfection, the culture medium was replaced with DMEM containing 10% FBS.

[0231] 48 hours after transfection, the expression of EGFP fluorescent protein indicated that the cells were successfully transfected, and the cells with positive EGFP expression were sorted for editing efficiency detection. The cells were subjected to genome extraction (using a genomic DNA extraction kit, TIANGEN, DP304-03). The PCR amplification product was used for high-throughput deep sequencing (Qingke Biotechnology Co., Ltd.) to detect the cleavage activity. The test results are shown in Figure 5 shown.

[0232] Analysis shows that each nuclease of the present invention has significant cleavage activity in mammalian cells and has potential application prospects.

[0233] The sequence information involved in the present invention is as follows:

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

Claims

1. A Cas protein, characterized in that The protein is selected from the group consisting of: (a) a polypeptide having an amino acid sequence as shown in any one of SEQ ID NOs: 1-4; (b) a polypeptide having ≥80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% homology (or identity) with the amino acid sequence shown in any one of SEQ ID NOs: 1-4, and the polypeptide has the biological function shown in any one of SEQ ID NOs: 1-4; (c) A derivative polypeptide formed by replacing, deleting or adding one or more (preferably 1-20, more preferably 1-10, and more preferably 1-5) amino acid residues of the amino acid sequence shown in any one of SEQ ID NOs: 1-4, and retaining the biological function shown in any one of SEQ ID NOs: 1-4.

2. A fusion protein comprising the Cas protein according to claim 1 and one or more functional domains.

3. A polynucleotide, characterized in that The polynucleotide is a nucleotide sequence encoding the Cas protein described in claim 1 or the fusion protein described in claim 2, or a nucleotide sequence encoding the fusion protein described in claim 3.

4. A CRISPR-Cas composition, comprising: (1) the Cas protein of claim 1 or the fusion protein of claim 2, or a nucleic acid molecule encoding the Cas protein of claim 1 or the fusion protein of claim 2; (2) guide RNA, or one or more DNA molecules encoding the guide RNA.

5. The CRISPR-Cas composition of claim 4, comprising one or more vectors comprising: (1) a first regulatory element, which is operably linked to a nucleotide sequence encoding the Cas protein or a nucleotide sequence encoding the fusion protein; and (2) a second regulatory element, wherein the second regulatory element is operably linked to a nucleotide sequence encoding the guide RNA, wherein the guide RNA comprises: (a) a spacer sequence capable of hybridizing to a target sequence of a target nucleic acid, and (b) a direct repeat (DR) sequence connected to the spacer sequence and capable of guiding the Cas protein to bind to the guide RNA to form a CRISPR-Cas complex targeting the target sequence; Wherein the first regulatory element and the second regulatory element are located on the same or different vectors of the CRISPR-Cas vector system.

6. A delivery system, characterized in that The delivery system comprises the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the CRISPR-Cas composition of any one of claims 4-5.

7. A host cell, characterized in that The host cell comprises the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the CRISPR-Cas composition of any one of claims 4-5, or the delivery system of claim 6.

8. A method for targeting and editing a target nucleic acid or cleaving a target nucleic acid, characterized in that: The method comprises contacting the target nucleic acid with the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the CRISPR-Cas composition of any one of claims 4-5, or the host cell of claim 6.

9. A method for non-specifically degrading or cleaving a single-stranded nucleic acid after recognizing a target nucleic acid, characterized in that: The method comprises contacting a target sequence of the target nucleic acid with the CRISPR-Cas composition of any one of claims 4-5.

10. A method for targeting and cleaving a double-stranded target nucleic acid, characterized in that: The method comprises contacting the double-stranded target nucleic acid with the CRISPR-Cas composition of any one of claims 4-5.

11. A method for inducing a change in cell state, characterized in that: The method comprises contacting the CRISPR-Cas composition of any one of claims 4-5 with the target nucleic acid in a cell.

12. A kit comprising the Cas protein of claim 1, the fusion protein of claim 2, or the polynucleotide of claim 3, or the CRISPR-Cas composition of any one of claims 4-5, or the host cell of claim 6, wherein the components of the kit are in the same or different containers.

13. The kit according to claim 12, which is used for one or more of gene or genome editing, disease treatment, targeting target genes, and cutting target genes or non-target genes.

14. A method for detecting a target nucleic acid in a sample, the method comprising contacting the sample with the CRISPR-Cas composition of any one of claims 4-5 and a non-target sequence; detecting a detectable signal generated by cleavage of the non-target sequence, thereby detecting the target nucleic acid; the non-target sequence does not hybridize with the guide RNA.

15. The Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the CRISPR-Cas composition of any one of claims 4 to 5, or the delivery system of claim 6, or the host cell of claim 7, or the use of claims 8 to 10 in nucleic acid detection.

16. Use of the Cas protein of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the CRISPR-Cas composition of any one of claims 4-5, or the delivery system of claim 6, or the host cell of claim 7 in the preparation of a medicament for treating a disorder or disease in a subject in need thereof.

17. The use according to claim 16, wherein the condition or disease comprises one or more of cystic fibrosis, pseudohypertrophic muscular dystrophy, Becker muscular dystrophy, alpha-1-antitrypsin deficiency, Pompe disease, myotonic dystrophy, Huntington's disease, fragile X syndrome, Friedreich's ataxia, amyotrophic lateral sclerosis, frontotemporal dementia, hereditary chronic kidney disease, hyperlipidemia, hypercholesterolemia, Leber's congenital amaurosis, sickle cell disease, hypercholesterolemia, transthyretin amyloidosis or beta-thalassemia.

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