CN114517190B - CRISPR enzymes and systems and uses - Google Patents

CRISPR enzymes and systems and uses Download PDF

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CN114517190B
CN114517190B CN202210112662.2A CN202210112662A CN114517190B CN 114517190 B CN114517190 B CN 114517190B CN 202210112662 A CN202210112662 A CN 202210112662A CN 114517190 B CN114517190 B CN 114517190B
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李珊珊
梁亚峰
赵庆芝
孙洁
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Shandong Shunfeng Biotechnology Co Ltd
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Abstract

The invention belongs to the field of nucleic acid editing, and particularly relates to the technical field of regularly clustered spaced short palindromic repeats (CRISPR). Specifically, the invention relates to a CRISPR enzyme and a system and application thereof, and particularly provides a novel Cas enzyme which has low homology with a reported Cas enzyme, can show nuclease activity in cells and outside the cells, and has wide application prospect.

Description

CRISPR enzymes and systems and uses
Technical Field
The invention relates to the field of gene editing, in particular to the technical field of regularly clustered spaced short palindromic repeats (CRISPR). In particular, the present invention relates to CRISPR enzymes and systems and uses, in particular to a novel CRISPR enzyme (alternatively referred to as CRISPR protein, cas effector protein, cas enzyme or Cas protein), fusion proteins comprising such proteins, and nucleic acid molecules encoding them. The invention also relates to complexes and compositions for nucleic acid editing (e.g., gene or genome editing) comprising a Cas protein or fusion protein of the invention, or a nucleic acid molecule encoding the same.
Background
The CRISPR/Cas technology is a widely used gene editing technology, which specifically binds to a target sequence on a genome and cleaves DNA to generate a double-strand break through RNA guide, and performs site-directed gene editing using biological non-homologous end joining or homologous recombination.
The CRISPR/Cas9 system is the most commonly used type II CRISPR system, which recognizes the PAM motif of 3' -NGG, performing blunt-end cleavage of the target sequence. The CRISPR/Cas Type V system is a newly discovered CRISPR system with a motif of 5' -TTN, which performs sticky end cleavage of target sequences, e.g. Cpf1, C2C1, casX, casY. However, the different CRISPRs/Cas currently available have different advantages and disadvantages. For example, cas9, C2C1 and CasX all require two RNAs for the guide RNA, whereas Cpf1 requires only one guide RNA and can be used for multiplex gene editing. CasX has a size of 980 amino acids, whereas the common Cas9, C2C1, casY and Cpf1 are typically around 1300 amino acids in size. In addition, the PAM sequences of Cas9, cpf1, casX, casY are all complex and diverse, and C2C1 recognizes the stringent 5' -TTN, so its target site is easily predicted than other systems, thereby reducing potential off-target effects.
In summary, given that currently available CRISPR/Cas systems are all limited by some drawbacks, the development of a new more robust CRISPR/Cas system with versatile good performance is of great significance for the development of biotechnology.
Disclosure of Invention
The inventors of the present application have unexpectedly discovered a novel endonuclease (Cas enzyme) through a large number of experiments and repeated trials. Based on this finding, the present inventors developed a novel CRISPR/Cas system, and a gene editing method and a nucleic acid detection method based on the system.
Cas effector protein
In one aspect, the invention provides a Cas protein that is an effector protein in a CRISPR/Cas system, also referred to herein as Cas-sf19 protein.
In one embodiment, the Cas protein amino acid sequence is identical to SEQ ID NO:1 compared to at least 80%, at least 85%, 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 and substantially retains the biological function of SEQ ID No. 1.
In one embodiment, the Cas protein and the Cas-sf19 protein of the present invention are derived from the same species.
In one embodiment, the Cas protein amino acid sequence is identical to SEQ ID NO:1, a sequence having substitution, deletion or addition of one or more amino acids; the one or more amino acids include substitutions, deletions or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
In one embodiment, the Cas protein of the present invention has any one or any several of the following functional domains i-iii:
i、PVSVMGIDLGVNPAFAYAVCT；
ii、KSYIDYYKNLRLDTLKKLTCAIVRTARSHGVEIVALEDIKRVDYDDQVKRAKENSLLSLWAPGMILERIEQELANEGIRTWRIDPRHTSQTACITDEFGY；
iii、GELLRVNSDVNAAINIARRFLTR。
it will be clear to those skilled in the art that the structure of a protein may be altered without adversely affecting its activity and functionality, for example, one or more conservative amino acid substitutions may be introduced into the amino acid sequence of a protein without adversely affecting the activity and/or three-dimensional structure of the protein molecule. Examples and embodiments of conservative amino acid substitutions will be apparent to those skilled in the art. Specifically, the amino acid residue may be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., a nonpolar amino acid residue is substituted for another nonpolar amino acid residue, a polar uncharged amino acid residue is substituted for another polar uncharged amino acid residue, a basic amino acid residue is substituted for another basic amino acid residue, and an acidic amino acid residue is substituted for another acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions where one amino acid is replaced by another amino acid belonging to the same group are within the scope of the present invention, as long as the substitution does not result in inactivation of the biological activity of the protein. Thus, the proteins of the invention may comprise one or more conservative substitutions in the amino acid sequence, which are preferably made by substitution according to Table 1. In addition, proteins that also comprise one or more other non-conservative substitutions are also encompassed by the present invention, provided that the non-conservative substitutions do not significantly affect the desired function and biological activity of the proteins of the present invention.
Conservative amino acid substitutions may be made at one or more predicted nonessential amino acid residues. A "nonessential" amino acid residue is an amino acid residue that can be altered (deleted, substituted, or substituted) without altering the biological activity, while an "essential" amino acid residue is required for biological activity. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Amino acid substitutions can be made in a non-conserved region of Cas-sf19. In general, such substitutions are not made to conserved amino acid residues, or to 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.
TABLE 1
Figure GDA0003843607420000031
Figure GDA0003843607420000041
It is well known in the art that one or more amino acid residues may be altered (substituted, deleted, truncated, or inserted) from the N-and/or C-terminus of a protein while still retaining its functional activity. Thus, proteins that have one or more amino acid residues altered from the N-and/or C-terminus of the Cas protein while retaining their 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, including PCR amplification by altering or extending the protein coding sequence by inclusion of amino acid coding sequences among the oligonucleotides used in PCR amplification.
It will be appreciated that proteins may be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions, and methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the Cas-sf19 protein can be made by mutation of the DNA. It may also be accomplished by other forms of mutagenesis and/or by directed evolution, e.g., using known methods of mutagenesis, recombination and/or shuffling (shuffling), in conjunction with related screening methods, to make single or multiple amino acid substitutions, deletions and/or insertions.
One skilled in the art will appreciate that these minor amino acid changes in the Cas-sf19 proteins of the present invention can occur (e.g., naturally occurring mutations) or be generated (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 change, but the polypeptide may retain its activity. Minor effects can be expected if the mutations present are not close to the catalytic domain, active site or other functional domains.
The essential amino acids of the Cas-sf19 protein can be identified by one skilled in the art according to methods known in the art, such as site-directed mutagenesis or protein evolution or analysis of biological information systems. The catalytic domain, active site or other functional domain of a protein can also be determined by physical analysis of the structure, such as by these techniques: such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations in putative key site amino acids.
In one embodiment, the Cas protein contains SEQ ID NO: 1.
In one embodiment, the Cas protein is SEQ ID NO: 1.
In one embodiment, the Cas protein is a protein that hybridizes to a polypeptide having the sequence of SEQ ID NO:1, and a derivative protein having the same biological function as the protein having the sequence shown in 1.
Such biological functions include, but are not limited to, binding to a guide RNA, endonuclease activity, binding to a specific site of a target sequence under the guidance of a guide RNA and cleavage activity, including, but not limited to Cis cleavage activity and Trans cleavage activity.
The invention also provides a fusion protein comprising a Cas protein as described above and other modifying moieties.
In one embodiment, the modifying moiety is selected from an additional protein or polypeptide, a detectable label, or any combination thereof.
In one embodiment, the modifying moiety is selected from an epitope tag, a reporter sequence, a Nuclear Localization Signal (NLS) sequence, a targeting moiety, a transcription activation domain (e.g., VP 64), a transcription repression domain (e.g., KRAB domain or SID domain), a nuclease domain (e.g., fok 1), and a domain having an activity selected from: nucleotide deaminase, methylase 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. Such NLS sequences are well known to those skilled in the art, and examples include, but are not limited to, the SV40 large T antigen, EGL-13, c-Myc, and TUS protein.
In one embodiment, the NLS sequence is located at, near, or near a terminus (e.g., N-terminus, C-terminus, or both) of a Cas protein of the invention.
Such epitope tags (epitoptags) are well known to those skilled in the art and include, but are not limited to, his, V5, FLAG, HA, myc, VSV-G, trx, etc., and other suitable epitope tags can be selected by those skilled in the art (e.g., purification, detection, or tracking).
The reporter gene sequences are well known to those skilled in the art, examples of which include, but are not limited to, GST, HRP, CAT, GFP, hcRed, dsRed, CFP, YFP, BFP, and the like.
In one embodiment, the fusion protein of the invention comprises a domain capable of binding to a DNA molecule or an intracellular molecule, such as Maltose Binding Protein (MBP), the DNA binding domain of Lex a (DBD), the DBD of GAL4, and the like.
In one embodiment, the fusion protein of the invention comprises a detectable label, such as a fluorescent dye, e.g. FITC or DAPI.
In one embodiment, the Cas protein of the present invention is coupled, conjugated or fused to the modifying moiety, optionally via a linker.
In one embodiment, the modification moiety is directly linked to the N-terminus or C-terminus of the Cas protein of the present invention.
In one embodiment, the modification moiety is linked to the N-terminus or C-terminus of the Cas protein of the present invention via a linker. Such linkers are well known in the art, examples of which include, but are not limited to, linkers comprising one or more (e.g., 1, 2, 3, 4, or 5) amino acids (e.g., glu or Ser) or amino acid derivatives (e.g., ahx, β -Ala, GABA, or Ava), or PEG, and the like.
The Cas protein, protein derivative or fusion protein of the present invention is not limited by the manner of its production, and for example, it may be produced by a genetic engineering method (recombinant technology) or may be produced by a chemical synthesis method.
Nucleic acid of Cas protein
In another aspect, the invention provides an isolated polynucleotide comprising:
(a) A polynucleotide sequence encoding a Cas protein or a fusion protein of the present invention;
(b) The sequence is shown as SEQ ID NO: 2;
(c) And SEQ ID NO:2 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base substitutions, deletions, or additions) compared to the sequence of (i);
(d) Nucleotide sequence and SEQ ID NO:2 (preferably 90% or more, more preferably 95% or more, most preferably 98%) and encodes a polypeptide of SEQ ID No. 1; or polynucleotide whose nucleotide sequence has homology of more than or equal to 80% (preferably more than or equal to 90%, more preferably more than or equal to 95%, most preferably more than or equal to 98%) with the sequence shown in SEQ ID NO. 2 and which encodes the polypeptide shown in SEQ ID NO. 1; or polynucleotide whose nucleotide sequence has homology of more than or equal to 80% (preferably more than or equal to 90%, more preferably more than or equal to 95%, most preferably more than or equal to 98%) with the sequence shown in SEQ ID NO. 2 and which encodes the polypeptide shown in SEQ ID NO. 1; alternatively, the first and second liquid crystal display panels may be,
(e) A polynucleotide complementary to any one of the polynucleotides of (a) - (d).
In one embodiment, the nucleotide sequence described in any of (a) - (e) is codon optimized for expression in a prokaryotic cell. In one embodiment, the nucleotide sequence described in any of (a) - (e) is codon optimized for expression in a eukaryotic cell.
In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.
Direct Repeat (Direct Repeat) sequences
In another aspect, the invention provides an engineered direct repeat that forms a complex with the Cas protein described above.
The direct repeat sequence is connected with a guide sequence capable of hybridizing with a target sequence to form a guide RNA (guide RNA or gRNA).
Hybridization of the target sequence to the gRNA represents at least 70%,75%,80%,85%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, or 100% identity of the target sequence and the nucleic acid sequence of the gRNA, such that a complex can be hybridized; or at least 12, 15, 16, 17, 18, 19, 20, 21, 22, or more bases of the nucleic acid sequences representing the target sequence and the gRNA can be complementarily paired to form a complex.
In some embodiments, the direct repeat sequence is identical to SEQ ID NO:3 have at least 90% sequence identity. In some embodiments, the direct repeat sequence is identical to SEQ ID NO:3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base substitutions, deletions, or additions) as compared to a sequence having one or more base substitutions, deletions, or additions.
In some embodiments, the direct repeat sequence is as set forth in SEQ ID NO:3, respectively.
Guide RNA (gRNA)
In another aspect, the present invention provides a gRNA comprising a first segment and a second segment; the first segment is also referred to as "framework region", "protein binding segment", "protein binding sequence", or "Direct Repeat (Direct Repeat) sequence"; the second segment is also referred to as a "targeting sequence for targeting nucleic acid" or a "targeting segment for targeting nucleic acid", or a "targeting sequence for targeting a target sequence".
The first segment of the gRNA is capable of interacting with a Cas protein of the invention, thereby allowing the Cas protein and the gRNA to form a complex.
The targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid of the present invention comprises a nucleotide sequence that is complementary to a sequence in the target nucleic acid. In other words, the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid of the present invention interacts with the target nucleic acid in a sequence-specific manner through hybridization (i.e., base pairing). Thus, the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid may be altered or modified to hybridize to any desired sequence within the target nucleic acid. The nucleic acid is selected from DNA or RNA.
The percent complementarity between the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid and the target sequence of the target nucleic acid can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).
The "framework region", "protein-binding segment", "protein-binding sequence", or "direct repeat" of a gRNA of the invention can interact with a CRISPR protein (or, cas protein). The gRNA of the invention directs its interacting Cas protein to a specific nucleotide sequence within a target nucleic acid through the action of a targeting sequence of the targeting nucleic acid.
Preferably, the guide RNA comprises a first segment and a second segment in the 5 'to 3' direction.
In the context of the present invention, the second segment is also understood to be a leader sequence which hybridizes to the target sequence.
The grnas of the invention are capable of forming a complex with the Cas protein.
The gRNA of the Cas protein of the present invention comprises a guide sequence that hybridizes to a target sequence, wherein the target sequence is located 3' of a Protospacer Adjacent Motif (PAM), which is 5' -ATG-3'.
Carrier
The present invention also provides a vector comprising a Cas protein, an isolated nucleic acid molecule or a polynucleotide as described above; preferably, it further comprises a regulatory element operably linked thereto.
In one embodiment, the regulatory element is selected from one or more of the group consisting of: enhancers, transposons, promoters, terminators, leader sequences, polyadenylation sequences, marker genes.
In one embodiment, the vector comprises a cloning vector, an expression vector, a shuttle vector, an integration vector.
In some embodiments, the vectors included in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), and may also be of the type of plasmids, viruses, cosmids, phages, and the like, which are well known to those skilled in the art.
Carrier system
The present invention provides an engineered, non-naturally occurring vector system, or CRISPR-Cas system, comprising a Cas protein or a nucleic acid sequence encoding said Cas protein and nucleic acid encoding one or more guide RNAs.
In one embodiment, the nucleic acid sequence encoding the Cas protein and the nucleic acid encoding the one or more guide RNAs are artificially synthesized.
In one embodiment, the nucleic acid sequence encoding the Cas protein and the nucleic acid encoding the one or more guide RNAs do not occur naturally together.
The one or more guide RNAs target one or more target sequences in the cell. The one or more target sequences hybridize to the genomic locus of the DNA molecule encoding the one or more gene products and direct the Cas protein to the genomic locus site of the DNA molecule of the one or more gene products, and the Cas protein modifies, edits, or cleaves the target sequence upon reaching the target sequence site, whereby expression of the one or more gene products is altered or modified.
The cells of the invention include one or more of animals, plants, or microorganisms.
In some embodiments, the Cas protein is codon optimized for expression in a cell.
In some embodiments, the Cas protein directs cleavage of one or both strands at the target sequence position.
The present invention also provides an engineered non-naturally occurring vector system, which may include one or more vectors, the one or more vectors including:
a) A first regulatory element operably linked to the gRNA,
b) A second regulatory element operably linked to the Cas protein;
wherein components (a) and (b) are located on the same or different carriers of the system.
The first and second regulatory elements include promoters (e.g., constitutive promoters or inducible promoters), enhancers (e.g., 35S promoter or 35S enhanced promoter), internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences).
In some embodiments, the vector in the system is a viral vector (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors and herpes simplex vectors), and may also be of the type of plasmid, virus, cosmid, phage, and the like, which are well known to those skilled in the art.
In some embodiments, the systems provided herein are in a delivery system. In some embodiments, the delivery system is a nanoparticle, a liposome, an exosome, a microbubble, and a gene gun.
In one embodiment, the target sequence is a DNA or RNA sequence from a prokaryotic or eukaryotic cell. In one embodiment, the target sequence is a non-naturally occurring DNA or RNA sequence.
In one embodiment, when the target sequence is DNA, the target sequence is located 3' of the pro-spacer adjacent motif (PAM), and the PAM has a sequence shown as 5' -ATG-3'.
In one embodiment, the target sequence is present within a cell. In one embodiment, the target sequence is present within the nucleus or within the cytoplasm (e.g., organelle). In one embodiment, the cell is a eukaryotic cell. In other embodiments, the cell is a prokaryotic cell.
In one embodiment, the Cas protein has one or more NLS sequences attached thereto. In one embodiment, the fusion protein comprises one or more NLS sequences. In one embodiment, the NLS sequence is linked to the N-terminus or C-terminus of the protein. In one embodiment, the NLS sequence is fused to the N-terminus or C-terminus of the protein.
In another aspect, the invention relates to an engineered CRISPR system comprising a Cas protein as described above and one or more guide RNAs, wherein the guide RNA comprises a direct repeat and a spacer sequence capable of hybridizing to a target nucleic acid, the Cas protein being capable of binding to the guide RNA and targeting a target nucleic acid sequence complementary to the spacer sequence.
Protein-nucleic acid complexes/compositions
In another aspect, the present invention provides a complex or composition comprising:
(i) A protein component selected from: the above Cas protein, derivatized protein, or fusion protein, and any combination thereof; and
(ii) A nucleic acid component comprising (a) a guide sequence capable of hybridizing to a target sequence; and (b) a direct repeat sequence capable of binding to a Cas protein of the present invention.
The protein component and the nucleic acid component are combined with each other to form a complex.
In one embodiment, the nucleic acid component is a guide RNA in a CRISPR-Cas system.
In one embodiment, the complex or composition is non-naturally occurring or modified. In one embodiment, at least one component of the complex or composition is non-naturally occurring or modified. In one embodiment, the first component is non-naturally occurring or modified; and/or, the second component is non-naturally occurring or modified.
Activated CRISPR complexes
In another aspect, the present invention also provides an activated CRISPR complex comprising: (1) a protein component selected from the group consisting of: a Cas protein, a derivatized protein, or a fusion protein of the invention, and any combination thereof; (2) A gRNA comprising (a) a guide sequence capable of hybridizing to a target sequence; and (b) a direct repeat sequence capable of binding to a Cas protein of the present invention; and (3) a target sequence that binds to the gRNA. Preferably, the binding is via a targeting sequence of a targeting nucleic acid on the gRNA to the target nucleic acid.
The terms "activated CRISPR complex", "activation complex" or "ternary complex" as used herein refer to a complex of a Cas protein, a gRNA, and a target nucleic acid in a CRISPR system after binding or modification.
The Cas protein and gRNA of the present invention can form a binary complex that is activated upon binding to a nucleic acid substrate that is complementary to a spacer sequence in the gRNA (alternatively referred to as a guide sequence that hybridizes to the target nucleic acid) to form an activated CRISPR complex. In some embodiments, the spacer sequence of the gRNA completely matches the target substrate. In other embodiments, the spacer sequence of the gRNA matches a portion (continuous or discontinuous) of the target substrate.
In a preferred embodiment, the activated CRISPR complex may exhibit a collateral nuclease activity, which refers to the non-specific or random cleavage activity of the activated CRISPR complex on single-stranded nucleic acids, also referred to in the art as trans cleavage activity.
Delivery and delivery compositions
The Cas proteins, grnas, fusion proteins, nucleic acid molecules, vectors, systems, complexes, and compositions of the invention can be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipofection, nuclear transfection, microinjection, sonoporation, gene gun, calcium phosphate-mediated transfection, cationic transfection, lipofection, dendritic transfection, heat shock transfection, nuclear transfection, magnetic transfection, lipofection, puncture transfection, optical transfection, agent-enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, viral particles, artificial virosomes, and the like.
Thus, in another aspect, the present invention provides a delivery composition comprising a delivery vehicle and one or any of the following: the Cas proteins, fusion proteins, nucleic acid molecules, vectors, systems, complexes and compositions of the present invention.
In one embodiment, the delivery vehicle is a particle.
In one embodiment, the delivery vector is selected from a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a microvesicle, a gene gun, or a viral vector (e.g., a replication defective retrovirus, lentivirus, adenovirus, or adeno-associated virus).
Host cell
The invention also relates to an in vitro, ex vivo or in vivo cell or cell line or progeny thereof comprising: cas proteins, fusion proteins, nucleic acid molecules, protein-nucleic acid complexes, activated CRISPR complexes, vectors, and delivery compositions of the invention described herein.
In certain embodiments, the cell is a prokaryotic cell.
In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a non-human mammalian cell, e.g., a non-human primate, bovine, ovine, porcine, canine, monkey, rabbit, rodent (e.g., rat or mouse) cell. In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a cell of a poultry bird (e.g., chicken), fish, or crustacean (e.g., clam, shrimp). In certain embodiments, the cell is a plant cell, e.g., a cell possessed by a monocot or dicot or a cell possessed by a cultivated plant or a food crop such as cassava, corn, sorghum, soybean, wheat, oat, or rice, e.g., an algae, a tree, or a producer, a fruit, or a vegetable (e.g., a tree such as a citrus tree, a nut tree; a solanum plant, cotton, tobacco, tomato, grape, coffee, cocoa, etc.).
In certain embodiments, the cell is a stem cell or stem cell line.
In certain instances, a host cell of the invention comprises a modification of a gene or genome that is not present in its wild type.
Gene editing method and application
The Cas protein, the nucleic acid, the composition as described above, the CIRSPR/Cas system as described above, the vector system as described above, the delivery composition as described above or the activated CRISPR complex as described above or the host cell as described above may be used for any one or several of the following uses: targeting and/or editing a target nucleic acid; cleaving double-stranded DNA, single-stranded DNA, or single-stranded RNA; non-specific cleavage and/or degradation of nucleic acid of lateral branches; non-specific cleavage of single-stranded nucleic acids; detecting nucleic acid; detecting nucleic acid in a target sample; specifically editing double-stranded nucleic acids; base-editing double-stranded nucleic acids; base-editing single-stranded nucleic acids. In other embodiments, the kit may also be used to prepare reagents or kits for any one or more of the uses described above.
The invention also provides the application of the Cas protein, the nucleic acid, the composition, the CIRCR SPR/Cas system, the vector system, the delivery composition or the activated CRISPR complex in gene editing, gene targeting or gene cutting; alternatively, use in the manufacture of a reagent or kit for gene editing, gene targeting or gene cleavage.
In one embodiment, the gene editing, gene targeting or gene cleavage is gene editing, gene targeting or gene cleavage inside and/or outside a cell.
The present invention also provides a method of editing, targeting or cleaving a target nucleic acid, comprising contacting the target nucleic acid with the Cas protein, nucleic acid, composition described above, CIRSPR/Cas system described above, vector system described above, delivery composition described above, or activated CRISPR complex described above. In one embodiment, the method is editing, targeting, or cleaving a target nucleic acid inside or outside the cell.
The gene editing or editing target nucleic acids include modifying genes, knocking out genes, altering expression of gene products, repairing mutations, and/or inserting polynucleotides, gene mutations.
The editing can be performed in prokaryotic cells and/or eukaryotic cells.
In another aspect, the invention also provides the application of the above Cas protein, nucleic acid, the above composition, the above CIRSPR/Cas system, the above vector system, the above delivery composition or the above activated CRISPR complex in nucleic acid detection, or in the preparation of a reagent or kit for nucleic acid detection.
In another aspect, the invention also provides a method of cleaving single-stranded nucleic acid, the method comprising contacting a nucleic acid population with the Cas protein and the grnas described above, wherein the nucleic acid population comprises a target nucleic acid and a plurality of non-target single-stranded nucleic acids, the Cas protein cleaving the plurality of non-target single-stranded nucleic acids.
The gRNA is capable of binding the Cas protein.
The gRNA is capable of targeting the target nucleic acid.
The contacting may be in vitro, ex vivo, or inside a cell in vivo.
Preferably, the cleaved single-stranded nucleic acid is non-specific cleavage.
In another aspect, the invention also provides the use of the above Cas protein, nucleic acid, the above composition, the above CIRSPR/Cas system, the above vector system, the above delivery composition or the above activated CRISPR complex for non-specific cleavage of single stranded nucleic acids, or for the preparation of a reagent or kit for non-specific cleavage of single stranded nucleic acids.
In another aspect, the invention also provides a kit for gene editing, gene targeting or gene cleavage, comprising the above Cas protein, gRNA, nucleic acid, the above composition, the above CIRSPR/Cas system, the above vector system, the above delivery composition, the above activated CRISPR complex, or the above host cell.
In another aspect, the present invention also provides a kit for detecting a target nucleic acid in a sample, the kit comprising: (ii) (a) a Cas protein, or a nucleic acid encoding the Cas protein; (b) A guide RNA, or a nucleic acid encoding the guide RNA, or a precursor RNA comprising the guide RNA, or a nucleic acid encoding the precursor RNA; and (c) a single stranded nucleic acid detector that is single stranded and does not hybridize to the guide RNA.
It is known in the art that precursor RNAs can be cleaved or processed into mature guide RNAs as described above.
In another aspect, the invention provides the use of the above Cas protein, nucleic acid, the above composition, the above CIRSPR/Cas system, the above vector system, the above delivery composition, the above activated CRISPR complex or the above host cell in the preparation of a formulation or kit for:
(i) Gene or genome editing;
(ii) Target nucleic acid detection and/or diagnosis;
(iii) Editing a target sequence in a target locus to modify an organism or non-human organism;
(iv) Treatment of disease;
(iv) Target genes are targeted.
Preferably, the gene or genome editing is gene or genome editing in a cell or outside the cell.
Preferably, the target nucleic acid detection and/or diagnosis is in vitro.
Preferably, the treatment of the disease is the treatment of a condition caused by a defect in the target sequence in the target locus.
In another aspect, the invention provides a method of detecting a target nucleic acid in a sample, the method comprising contacting the sample with the Cas protein, a gRNA (guide RNA) comprising a region that binds to the Cas protein and a guide sequence that hybridizes to the target nucleic acid, and a single-stranded nucleic acid detector; detecting a detectable signal generated by the Cas protein-cleaved single-stranded nucleic acid detector, thereby detecting a target nucleic acid; the single-stranded nucleic acid detector does not hybridize to the gRNA.
Method for specifically modifying target nucleic acid
In another aspect, the present invention also provides a method of specifically modifying a target nucleic acid, the method comprising: contacting the target nucleic acid with the Cas protein, the nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition, or the activated CRISPR complex.
The specific modification may occur in vivo or in vitro.
The specific modification may occur intracellularly or extracellularly.
In some cases, the cell is selected from a prokaryotic cell or a eukaryotic cell, e.g., an animal cell, a plant cell, or a microbial cell.
In one embodiment, the modification refers to a break in the target sequence, e.g., a single/double strand break in DNA, or a single strand break in RNA.
In some cases, the method further comprises contacting the target nucleic acid with a donor polynucleotide, wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of the copy of the donor polynucleotide is integrated into the target nucleic acid.
In one embodiment, the modification further comprises inserting an editing template (e.g., an exogenous nucleic acid) into the break.
In one embodiment, the method further comprises: contacting the editing template with the target nucleic acid, or delivering into a cell comprising the target nucleic acid. In this embodiment, the method repairs the disrupted target gene by homologous recombination with an exogenous template polynucleotide; in some embodiments, the repair results in a mutation, including an insertion, deletion, or substitution of one or more nucleotides of the target gene, and in other embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.
Detection (non-specific cleavage)
In another aspect, the invention provides a method of detecting a target nucleic acid in a sample, the method comprising contacting the sample with the above-described Cas protein, nucleic acid, the above-described composition, the above-described CIRSPR/Cas system, the above-described vector system, the above-described delivery composition, or the above-described activated CRISPR complex, and a single-stranded nucleic acid detector; detecting a detectable signal generated by the Cas protein cleavage single stranded nucleic acid detector, thereby detecting the target nucleic acid.
In the present invention, the target nucleic acid includes a ribonucleotide or a deoxyribonucleotide; including single-stranded nucleic acids, double-stranded nucleic acids, such as single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA.
In one embodiment, the target nucleic acid is derived from a sample of a virus, bacterium, microorganism, soil, water source, human, animal, plant, or the like. Preferably, the target nucleic acid is a product enriched or amplified by PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM and other methods.
In one embodiment, the target nucleic acid is a viral nucleic acid, a bacterial nucleic acid, a specific nucleic acid associated with a disease, such as a specific mutation site or SNP site, or a nucleic acid that is different from a control; preferably, the virus is a plant virus or an animal virus, e.g., papilloma virus, hepatic DNA virus, herpes virus, adenovirus, poxvirus, parvovirus, coronavirus; preferably, the virus is a coronavirus, preferably SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, mers-CoV.
In the present invention, the gRNA is matched to a target sequence on a target nucleic acid by at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.
In one embodiment, when the target sequence contains one or more characteristic sites (e.g., a particular mutation site or SNP), the characteristic site completely matches the gRNA.
In one embodiment, one or more grnas with different targeting sequences may be included in the detection method, targeting different target sequences.
In the present invention, the single-stranded nucleic acid detector includes, but is not limited to, single-stranded DNA, single-stranded RNA, DNA-RNA hybrid, nucleic acid analogs, base modifications, and single-stranded nucleic acid detectors containing abasic spacers; "nucleic acid analogs" include, but are not limited to: locked nucleic acids, bridged nucleic acids, morpholino nucleic acids, ethylene glycol nucleic acids, hexitol nucleic acids, threose nucleic acids, arabinose nucleic acids, 2' oxymethyl RNA, 2' methoxyacetyl RNA, 2' fluoro RNA, 2' amino RNA, 4' thio RNA, and combinations thereof, including optional ribonucleotide or deoxyribonucleotide residues.
In the present invention, the detectable signal is realized by: vision-based detection, sensor-based detection, color detection, fluorescence signal-based detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase transition/dispersion, electrochemical detection, and semiconductor-based detection.
In the present invention, it is preferable that a fluorescent group and a quencher group are respectively disposed at both ends of the single-stranded nucleic acid detector, and when the single-stranded nucleic acid detector is cleaved, a detectable fluorescent signal can be exhibited. The fluorescent group is selected from one or more of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, texas Red or LC RED 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, dabcy1 or Tamra.
In other embodiments, different labeled molecules are respectively disposed at the 5 'end and the 3' end of the single-stranded nucleic acid detector, and the results of the colloidal gold test before and after cleavage by the Cas protein of the single-stranded nucleic acid detector are detected by means of colloidal gold detection; the single-stranded nucleic acid detector shows different color development results on a colloidal gold detection line and a quality control line before and after being cut by the Cas protein.
In some embodiments, the method of detecting a target nucleic acid can further comprise comparing the level of the detectable signal to a reference signal level, and determining the amount of the target nucleic acid in the sample based on the level of the detectable signal.
In some embodiments, the method of detecting a target nucleic acid can further comprise using an RNA reporter nucleic acid and a DNA reporter nucleic acid (e.g., fluorescent color) on different channels and determining the level of detectable signal by measuring the signal levels of the RNA and DNA reporter molecules and by measuring the amount of target nucleic acid in the RNA and DNA reporter molecules, sampling based on combining (e.g., using a minimum or product) the levels of detectable signal.
In one embodiment, the target gene is present in a cell.
In one embodiment, the cell is a prokaryotic cell.
In one embodiment, the cell is a eukaryotic cell.
In one embodiment, the cell is an animal cell.
In one embodiment, the cell is a human cell.
In one embodiment, the cell is a plant cell, such as a cell possessed by a cultivated plant (e.g., cassava, corn, sorghum, wheat, or rice), an algae, a tree, or a vegetable.
In one embodiment, the target gene is present in a nucleic acid molecule (e.g., a plasmid) in vitro.
In one embodiment, the target gene is present in a plasmid.
Definition of terms
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the procedures of molecular genetics, nucleic acid chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.
Cas protein
In the present invention, cas protein, cas enzyme, cas effector protein may be used interchangeably; the present inventors have for the first time discovered and identified a Cas effector protein having an amino acid sequence selected from the group consisting of:
(i) SEQ ID NO: 1;
(ii) And SEQ ID NO:1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions) compared to the sequence of (a); or
(iii) And SEQ ID NO:1, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, 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.
Nucleic acid cleavage or cleavage of nucleic acids herein includes DNA or RNA fragmentation in a target nucleic acid (Cis cleavage), DNA or RNA fragmentation in a side-branch nucleic acid substrate (single-stranded nucleic acid substrate) (i.e., non-specific or non-targeting, trans cleavage) produced by a Cas enzyme as described herein. 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.
CRISPR system
As used herein, the terms "regularly clustered short palindromic repeats (CRISPR) -CRISPR-associated (Cas) (CRISPR-Cas) system" or "CRISPR system" are used interchangeably and have the meaning generally understood by those skilled in the art, which generally comprise a transcript or other element that is associated with the expression of a CRISPR-associated ("Cas") gene, or a transcript or other element that is capable of directing the activity of said Cas gene.
CRISPR/Cas complexes
As used herein, the term "CRISPR/Cas complex" refers to a complex formed by the binding of a guide RNA (guide RNA) or mature crRNA to a Cas protein, which comprises a direct repeat that hybridizes to a guide sequence of a target sequence and binds to the Cas protein, which complex is capable of recognizing and cleaving a polynucleotide that is capable of hybridizing to the guide RNA or mature crRNA.
Guide RNA (guide RNA, gRNA)
As used herein, the terms "guide RNA", "gRNA", "mature crRNA", "guide sequence" are used interchangeably and have the meaning commonly understood by those skilled in the art. In general, the guide RNA may comprise, consist essentially of, or consist of a direct repeat (direct repeat) and a guide sequence.
In certain instances, the guide sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence to hybridize to the target sequence and direct specific binding of the CRISPR/Cas complex to the target sequence. In one embodiment, the degree of complementarity between a 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 optimally aligned. Determining the optimal alignment is within the ability of one of ordinary skill in the art. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, clustalW, the Smith-Waterman algorithm in matlab (Smith-Waterman), bowtie, geneius, biopython, and SeqMan.
Target sequence
By "target sequence" is meant a polynucleotide that is targeted by a guide sequence in the gRNA, e.g., a sequence that is complementary to the guide sequence, wherein hybridization between the target sequence and the guide sequence will promote formation of a CRISPR/Cas complex (including Cas protein and gRNA). Complete complementarity is not necessary as long as there is sufficient complementarity to cause hybridization and promote formation of a CRISPR/Cas complex.
The target sequence may comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located intracellularly or extracellularly. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence may be located within an organelle of the eukaryotic cell, such as a mitochondrion or chloroplast. Sequences or templates that can be used for recombination into a target locus containing the target sequence are referred to as "editing templates" or "editing polynucleotides" or "editing sequences". In one embodiment, the editing template is an exogenous nucleic acid. In one embodiment, the recombination is homologous recombination.
In the present invention, a "target sequence" or "target polynucleotide" or "target nucleic acid" can be any polynucleotide endogenous or exogenous to a cell (e.g., a eukaryotic cell). For example, the target polynucleotide may 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 should be related to the Protospacer Adjacent Motif (PAM).
Single-stranded nucleic acid detector
The single-stranded nucleic acid detector of the present invention refers to a sequence containing 2 to 200 nucleotides, preferably, 2 to 150 nucleotides, preferably, 3 to 100 nucleotides, preferably, 3 to 30 nucleotides, preferably, 4 to 20 nucleotides, and more preferably, 5 to 15 nucleotides. Preferably a single-stranded DNA molecule, a single-stranded RNA molecule or a single-stranded DNA-RNA hybrid.
The single-stranded nucleic acid detector comprises different reporter groups or marker molecules at both ends, and does not present a reporter signal when in an initial state (i.e., an uncleaved state), and presents a detectable signal when the single-stranded nucleic acid detector is cleaved, i.e., presents a detectable difference after cleavage from before cleavage.
In one embodiment, the reporter group or the marker molecule comprises a fluorescent group and a quenching group, wherein the fluorescent group is selected from one or any several of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, texas Red or LC RED 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, dabcy1 or Tamra.
In one embodiment, the single stranded nucleic acid detector has a first molecule (e.g., FAM or FITC) attached to the 5 'end and a second molecule (e.g., biotin) attached to the 3' end. The reaction system containing the single-stranded nucleic acid detector is used in combination with a flow strip to detect the target nucleic acid (preferably, in a colloidal gold detection manner). The flow strip is designed with two capture lines, with an antibody that binds to a first molecule (i.e. a first molecular antibody) at the sample contacting end (colloidal gold), an antibody that binds to the first molecular antibody at the first line (control line), and an antibody that binds to a second molecule (i.e. a second molecular antibody, such as avidin) at the second line (test line). As the reaction flows along the strip, the first molecular antibody binds to the first molecule carrying the cleaved or uncleaved oligonucleotide to the capture line, the cleaved reporter will bind to the antibody of the first molecular antibody at the first capture line, and the uncleaved reporter will bind to the second molecular antibody at the second capture line. Binding of the reporter group at each line will result in a strong readout/signal (e.g. color). As more reporters are cut, more signal will accumulate at the first capture line and less signal will appear at the second line. In certain aspects, the invention relates to the use of a flow strip as described herein for detecting nucleic acids. In certain aspects, the invention relates to a method of detecting nucleic acids using a flow strip as defined herein, e.g. a (side) flow test or a (side) flow immunochromatographic assay. In some aspects, the molecules in the single-stranded nucleic acid detector may be replaced with each other, or the positions of the molecules may be changed, and the modified form is also included in the present invention as long as the reporting principle is the same as or similar to that of the present invention.
The detection method of the present invention can be used for quantitative detection of a target nucleic acid to be detected. The quantitative detection index can be quantified according to the signal intensity of the reporter group, such as the luminous intensity of a fluorescent group, or the width of a color development strip.
Wild type
As used herein, the term "wild-type" has the meaning commonly understood by those skilled in the art to mean a typical form of an organism, strain, gene, or characteristic that distinguishes it from a mutant or variant form when it exists in nature, which may be isolated from a source in nature and which has not been intentionally modified by man.
Derivatization
As used herein, the term "derivatize" refers to a chemical modification of an amino acid, polypeptide, or protein to which one or more substituents have been covalently attached. The substituents may also be referred to as side chains.
The derivatized protein is a derivative of the protein, and generally, derivatization of the protein does not adversely affect the desired activity of the protein (e.g., activity in binding to a guide RNA, endonuclease activity, activity in binding to and cleaving at a particular site in a target sequence under the guidance of the guide RNA), i.e., the derivative of the protein has the same activity as the protein.
Derivatized proteins
Also referred to as "protein derivatives" refer to modified forms of proteins, for example, wherein one or more amino acids of the protein may be deleted, inserted, modified and/or substituted.
Not naturally occurring
As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and represent artificial participation. When these terms are used to describe a nucleic acid molecule or polypeptide, it means that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component with which it is associated in nature or as found in nature.
Orthologues (orthologues)
As used herein, the term "ortholog" has the meaning commonly understood by those skilled in the art. By way of further guidance, "ortholog" of a protein as described herein refers to a protein belonging to a different species that performs the same or similar function as the protein as its ortholog.
Identity of each other
As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (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), then the molecules are identical at that position. The "percent 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 x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignments can be performed by using, for example, needleman et al (1970) j.mol.biol.48: 443-453. The algorithm of e.meyers and w.miller (comput.appl biosci.,4, 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a gap penalty of 4. In addition, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithms that have been incorporated into the GAP program of the GCG software package (available at www. GCG. Com), using either the Blossum 62 matrix or the PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.
Carrier
The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and other diverse polynucleotides known in the art. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. A vector can be introduced into a host cell to thereby produce a transcript, protein, or peptide, including from a protein, fusion protein, isolated nucleic acid molecule, etc. (e.g., a CRISPR transcript, such as a nucleic acid transcript, protein, or enzyme) as described herein. A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication origin.
One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques.
Another type of vector is a viral vector, wherein the virus-derived DNA or RNA sequences are present in the vector for packaging of viruses (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses). Viral vectors also comprise polynucleotides carried by viruses for transfection into a host cell. Certain vectors (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced.
Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors".
Host cell
As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, eukaryotic cells such as microbial cells, fungal cells, animal cells, and plant cells.
One skilled in the art will appreciate that the design of an expression vector may depend on factors such as the choice of host cell to be transformed, the level of expression desired, and the like.
Regulatory element
As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly U sequences), which are described in detail with reference to gordel (Goeddel), "gene expression technology: METHODS IN ENZYMOLOGY (GENE EXPRESSION TECHNOLOGY: METHOD IN ENZYMOLOGY) 185, academic Press, san Diego, calif. (1990). In some cases, regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells as well as those sequences that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may primarily direct expression in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocyte). In certain instances, the regulatory element may also direct expression in a time-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue or cell type specific. In certain instances, the term "regulatory element" encompasses enhancer elements, such as WPRE; a CMV enhancer; the R-U5' fragment in the LTR of HTLV-I ((mol. Cell. Biol., vol.8 (1), pp.466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β -globin (Proc. Natl. Acad. Sci. USA., vol.78 (3), pp.1527-31, 1981).
Promoters
As used herein, the term "promoter" has a meaning well known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of a gene that is capable of promoting expression of a downstream gene. Constitutive (constitutive) promoters are nucleotide sequences that: when operably linked to a polynucleotide that encodes or defines a gene product, it results in the production of the gene product in the cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or defines a gene product, causes the gene product to be produced intracellularly substantially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that: when operably linked to a polynucleotide that encodes or defines a gene product, it results in the production of the gene product in the cell substantially only if the cell is of the tissue type to which the promoter corresponds.
NLS
A "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" a protein for introduction into the nucleus by nuclear transport, i.e., a protein with NLS is transported to the nucleus. Typically, NLS contains positively charged Lys or Arg residues exposed on the surface of the protein. Exemplary nuclear localization sequences include, but are not limited to, NLS from: SV40 Large T antigen, EGL-13, c-Myc and TUS protein. In some embodiments, the NLS comprises a PKKKRKV sequence. In some embodiments, the NLS comprises the avkrpaatkkagqakkblld sequence. In some embodiments, the NLS comprises a PAAKRVKLD sequence. In some embodiments, the NLS comprises the sequence MSRRRKANPTKLSENAKKLAKEVEN. In some embodiments, the NLS comprises a KLKIKRPVK sequence. Other nuclear localization sequences include, but are not limited to, the acidic M9 domain of hnRNP A1, the sequence KIPIK in yeast transcriptional repressor Mat α 2, and PY-NLS.
Is operably connected to
As used herein, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
Complementarity
As used herein, the term "complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by means of a conventional watson-crick or other unconventional type. Percent complementarity refers to the percentage of residues (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary) in a nucleic acid molecule that can form hydrogen bonds (e.g., watson-crick base pairing) with a second nucleic acid sequence. "completely complementary" means that all consecutive residues of one nucleic acid sequence hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "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.
Stringent conditions
As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are generally sequence dependent and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.
Hybridization of
The terms "hybridize" or "complementary" or "substantially complementary" refer to a nucleic acid (e.g., RNA, DNA) that comprises a nucleotide sequence that enables it to bind non-covalently, i.e., to form base pairs and/or G/U base pairs with another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid binds specifically to the complementary nucleic acid), "anneal" or "hybridize".
Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. Suitable conditions for hybridization between two nucleic acids depend on the length and degree of complementarity of the nucleic acids, variables well known in the art. Typically, the length of the hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).
It is understood that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to specifically hybridize. A polynucleotide may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or a target region that hybridizes thereto has 100% sequence complementarity of the target region.
Hybridization of a target sequence to a gRNA represents that at least 60%, 70%,75%,80%,85%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, or 100% of the target sequence and the nucleic acid sequence of the gRNA can hybridize to form a complex; or at least 12, 15, 16, 17, 18, 19, 20, 21, 22 or more bases of nucleic acid sequences representing the target sequence and the gRNA can be complementarily paired to hybridize to form a complex.
Expression of
As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and/or the process by which transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.
Joint
As used herein, the term "linker" refers to a linear polypeptide formed from a plurality of amino acid residues joined by peptide bonds. The linker of the present invention may be an artificially synthesized amino acid sequence, or a naturally occurring polypeptide sequence, such as a polypeptide having a hinge region function. Such linker polypeptides are well known in the art (see, e.g., holliger, P. Et al (1993) Proc. Natl. Acad. Sci. USA 90 6444-6448 Poljak, R.J. Et al (1994) Structure 2.
Treatment of
As used herein, the term "treating" refers to treating or curing a disorder, delaying the onset of symptoms of a disorder, and/or delaying the development of a disorder.
Subject of the disease
As used herein, the term "subject" includes, but is not limited to, various animals, plants, and microorganisms.
Animal(s) production
For example, a mammal, such as a bovine, equine, ovine, porcine, canine, feline, lagomorph, rodent (e.g., mouse or rat), non-human primate (e.g., macaque or cynomolgus monkey), or human. In certain embodiments, the subject (e.g., human) has a disorder (e.g., a disorder resulting from a deficiency in a disease-associated gene).
Plant and method for producing the same
The term "plant" is to be understood as meaning any differentiated multicellular organism capable of photosynthesis, in the context of including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops, including artichokes, corm cabbages, arugula, leeks, asparagus, lettuce (e.g. head lettuce, leaf lettuce, longifola lettuce), pakchoi (bok choy), russiae, melons (e.g. melons, watermelons, crow melon (crenshaw), honeydew melon, cantaloupe), rape crops (e.g., cabbage, cauliflower, broccoli, kale, chinese cabbage, pakchoi, cabbages), artichoke, carrot, cabbage (napa), okra, onion, celery, parsley, chickpea, parsnip, chicory, pepper, potato, cucurbits (e.g., zucchini, cucumber, courgette, squash, pumpkin), radish, onion, turnip cabbage, purple eggplant (also known as eggplant), salsify, endive, shallot, endive, garlic, spinach, green onion, squash, greengrophytes (greens), beets (sugarbeet and fodder beet), sweet potato, swiss chard, horseradish, tomato, turnip, and spices; fruit and/or vine crops such as apple, apricot, cherry, nectarine, peach, pear, plum, prune, cherry, quince, almond, chestnut, hazelnut, pecan, pistachio, walnut, citrus, blueberry, boysenberry (boysenberry), raspberry, currant, loganberry, raspberry, strawberry, blackberry, grape, avocado, banana, kiwi, persimmon, pomegranate, pineapple, tropical fruit, pome, melon, mango, papaya, and lychee; field crops, such as clover, alfalfa, evening primrose, meadowfoam, corn/maize (fodder corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain crops (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, leguminous plants (beans, lentils, peas, soybeans), oil-bearing plants (rape, mustard, olives, sunflowers, coconut, castor oil plants, cocoa beans, groundnuts), arabidopsis, fiber plants (cotton, flax, jute), lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plants; and/or bedding plants, such as flowering plants, cactus, fleshy plants and/or ornamental plants, and trees, such as forests (broad leaf and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other plantlets.
Advantageous effects of the invention
The invention discovers a novel Cas enzyme, and Blast results show that the Cas enzyme has low consistency with the reported Cas enzyme, can show the activity of nuclease in vivo and in vitro, and has wide application prospect.
Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.
Drawings
FIG. 1 prediction of Cas-sf19 active domain.
Figure 2-fluorescence results of cas-sf19 for nucleic acid detection.
PAM identification of Cas-sf19.
Figure 4 fluorescence results of cas-sf19 for double stranded target nucleic acid detection.
FIG. 5 is a graph showing the cleavage result of Cas-sf19 for double-stranded nucleic acids.
Sequence information
SEQ ID NO: Description of the invention
1 Amino acid sequence of Cas-sf19
2 Cas-sf19 nucleic acid sequence
3 DR region of gRNA of Cas-sf19
Detailed Description
The following examples are intended to illustrate the invention only and are not intended to limit the invention. Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention can be found in Sambrook (Sambrook), friesch (Fritsch), and manitis (manitis), molecular cloning: a LABORATORY Manual (Molecular CLONING: A Laboratory Manual), 2 nd edition (1989); current MOLECULAR BIOLOGY laboratory Manual (Current Protocols IN MOLECULAR BIOLOGY) (edited by F.M. Otsubel et al, (1987)); METHODS IN ENZYMOLOGY (METHODS IN Enzymology) series (academic Press): PCR 2: PRACTICAL methods (PCR 2: a LABORATORY Manual (ANTIBODIES, A LABORATORY MANUAL), and ANIMAL CELL CULTURE (ANIMAL CELL CULTURE) (edited by R.I. Freyrnib (R.I. Freshney) (1987)).
In addition, those whose specific conditions are not specified in the examples are conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. It will be appreciated by those skilled in the art that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.
Example 1 acquisition of Cas protein
The inventor analyzes the macro genome of the uncultured substance, and identifies and obtains a new Cas enzyme through redundancy removal and protein clustering analysis, wherein the amino acid sequence of the new Cas enzyme is shown as SEQ ID NO:1, and the nucleic acid sequence is shown as SEQ ID NO:2, respectively. Blast results show that the Cas protein has low sequence identity with the reported Cas protein, and the Cas protein is named Cas-sf19 in the invention.
Analysis shows that the homologous repeat sequence of gRNA corresponding to the Cas-sf19 protein is GUGCUGCCGGUCUAAUCGGGGAUCGGAAUUGCAC.
The Cas-sf19 protein comprises 911 amino acids, and is analyzed in comparison with the structural domains in a Pfam database, and the analysis result is shown in FIG. 1; three functional domains, ruvC _1 (PF 18516) RuvC nuclear domain, with endoribonuclease activity, are predicted, with the following information for 3 RuvC nuclear domains in the Cas-sf19 protein sequence:
(1)472-492：PVSVMGIDLGVNPAFAYAVCT；
(2)664-763：KSYIDYYKNLRLDTLKKLTCAIVRTARSHGVEIVALEDIKRVDYDDQVKRAKENSLLSLWAPGMILERIEQELANEGIRTWRIDPRHTSQTACITDEFGY；
(3)778-800：GELLRVNSDVNAAINIARRFLTR。
example 2 application of Cas-sf19 protein in nucleic acid detection
This example was tested in vitro to verify the trans cleavage activity of Cas-sf19. The gRNA which can be paired with the target nucleic acid is used for guiding the Cas-sf19 protein to be recognized and combined on the target nucleic acid in the embodiment; subsequently, the Cas-sf19 protein activates trans cleavage activity on any single-stranded nucleic acid, thereby cleaving the single-stranded nucleic acid detector in the system; the two ends of the single-stranded nucleic acid detector are respectively provided with a fluorescent group and a quenching group, and if the single-stranded nucleic acid detector is cut, fluorescence can be excited; in other embodiments, both ends of the single-stranded nucleic acid detector may be provided with a label capable of being detected by colloidal gold.
In this example, the target nucleic acid was selected to be a single-stranded DNA, N-B-i3g1-ssDNA0, having the sequence: CGACATTCCGAAGAACGCGCGCTGGAATTGGCGGC;
the gRNA sequence is GUGCUGCCGGUCUCUAAUCGGGGAUCGGAAUUGCACCCCCCAGCGCUUCAGCGUUC(the underlined region is the targeting region);
the single-stranded nucleic acid detector sequence is FAM-TTGTT-BHQ1;
the following reaction system is adopted: cas-sf19 final concentration is 50nM, gRNA final concentration is 50nM, target nucleic acid final concentration is 50nM, single stranded nucleic acid detector final concentration is 200nM. Incubation at 37 ℃ and reading FAM fluorescence/1 min. The control group had no target nucleic acid added.
As shown in fig. 2, compared to the control without the target nucleic acid, the single-stranded nucleic acid in the Cas-sf19 cleavage system is detected in the presence of the target nucleic acid, and fluorescence is rapidly reported. The above experiments reflect that Cas-sf19 can be used for detection of target nucleic acids in conjunction with a single-stranded nucleic acid detector. In FIG. 2, 1 is the result of the experiment with the target nucleic acid added, and 2 is the control group without the target nucleic acid added. In addition, other single-stranded nucleic acid detectors, such as 5'6-FAM/TTATT/3' BHQ1, cas-sf19, can also show good cleavage activity and can be used for in vitro nucleic acid detection.
Example 3 PAM Domain identification of Cas-sf19 protein
Constructing a Cas-sf19 protein expression plasmid: after the codon optimization of the nucleic acid sequence of Escherichia coli, gene synthesis is carried out, and the Escherichia coli expression vector PeT28 (a) + vector is connected. Adding JM23119 promoter into a PeT28 (a) + -Cas-sf19 protein vector to start the transcription of Cas-sf19 CrRNA. Forming a carrier: peT28 (a) + -Cas-sf19-JM23119-crRNA; construction of PAM library: <xnotran> CGTGTTTCGTAAAGTCTGGAAACGCGGAAGCCCCCAGCGCTTCAGCGTTCNNNNNNTCCCCTACGTGCTGCTGAAGTTGCCCGCAA, N . </xnotran> After filling in with Klenow enzyme, the vector pacyc184 was ligated. After transforming the Escherichia coli, extracting plasmids to form a PAM library.
PAM library subtraction experiment: preparing competence: BL21 (DE 3) -PeT28 (a) + -Cas-sf19-JM23119-crRNA. PAM library plasmid transformation competence: BL21 (DE 3) -PeT28 (a) + -Cas-sf19-JM23119-crRNA is coated on an LB plate containing kanamycin and chloramphenicol, cultured overnight at 37 ℃, thallus is collected, and a large-extraction kit is used for plasmid extraction to obtain a post-subtraction PAM library. And carrying out PCR reaction by taking 30 ng/mu L of plasmid (PAM library) as a template primer to obtain a control group sample, and carrying out PCR reaction by taking 30 ng/mu L of plasmid (PAM library after subtraction) as a template to obtain an experimental group sample. And (4) sending the control group samples and the experimental group samples to second-generation sequencing for data analysis.
Obtaining a PAM structure for Cas-sf 19: the number of occurrences in the experimental and control groups was counted separately for 4096 PAM sequences and normalized by the total number of all PAM sequences in each group. The consumption level for each PAM was calculated as log2 (control group normalized value/experimental group normalized value), and when this value was greater than 3.5, this PAM was considered to be significantly consumed. The significantly depleted PAM sequence was then predicted using Weblogo and found to be ATG in PAM structure, as shown in figure 3.
Example 4 application of Cas-sf19 protein in detection of double-stranded target nucleic acid
This example demonstrates in vitro assay for Cas-sf 19-induced trans cleavage activity in vitro targeting double-stranded DNA. The gRNA which can be paired with the target nucleic acid is used for guiding the Cas-sf19 protein to be recognized and combined on the target nucleic acid in the embodiment; subsequently, the Cas-sf19 protein activates trans cleavage activity on any single-stranded nucleic acid, thereby cleaving the single-stranded nucleic acid detector in the system; the two ends of the single-stranded nucleic acid detector are respectively provided with a fluorescent group and a quenching group, and if the single-stranded nucleic acid detector is cut, fluorescence can be excited; in other embodiments, both ends of the single-stranded nucleic acid detector may be provided with a label capable of being detected by colloidal gold.
In this example, the target nucleic acid was selected to be a double-stranded DNA (plasmid), T-N-B-ATG, whose sequence was:
Figure GDA0003843607420000281
Figure GDA0003843607420000282
Figure GDA0003843607420000283
connecting a Vector T-Vector-pEASY-Blunt Simple Cloning Vector; the italicized portion (ATG) is the PAM sequence and the underlined region is the targeting region.
The gRNA sequence is GUGCUGCCGGUCUCUAAUCGGGGAUCGGAAUUGCACCCCCCAGCGCUUCAGCGUUC(the underlined region is the targeting region);
the single-stranded nucleic acid detector sequence is FAM-TTATT-BHQ1;
the following reaction system is adopted: cas-sf19 final concentration is 100nM, gRNA final concentration is 50nM, double-stranded target nucleic acid final concentration is 5 ng/. Mu.L, single-stranded nucleic acid detector final concentration is 200nM. Incubation at 37 ℃ and reading FAM fluorescence/30 s.
As a result, as shown in FIG. 4, compared to the control without the target nucleic acid, the single-stranded nucleic acid in the Cas-sf19 cleavage system was detected in the presence of the target nucleic acid, and fluorescence was rapidly reported. The above experiments reflect that Cas-sf19 can be used for detection of double stranded target nucleic acids in conjunction with a single stranded nucleic acid detector. In FIG. 4, 1 is the result of the experiment in which the double-stranded target nucleic acid was added, and 2 is the control group in which the double-stranded target nucleic acid was not added.
Example 5 use of Cas-sf19 protein for nucleic acid editing
This example measures cis cleavage activity of double stranded DNA of Cas-sf19. In this example, the Cas-sf19 protein is guided by gRNA that can pair with the target nucleic acid to recognize and bind to the target nucleic acid, thereby cleaving the target nucleic acid in the system, and the cleaved target nucleic acid is subjected to agarose electrophoresis detection.
In this example, the target nucleic acid was selected to be a double-stranded DNA (plasmid), T-N-B-ATG, whose sequence was:
Figure GDA0003843607420000291
Figure GDA0003843607420000292
Figure GDA0003843607420000293
connecting a Vector T-Vector-pEASY-Blunt Simple Cloning Vector; the italic part is the PAM sequence and the underlined region is the targeting region. The gRNA sequence is GUGCUGCCGGUCUCUAAUCGGGGAUCGGAAUUGCACCCCCCAGCGCUUCAGCGUUC(the underlined region is the targeting region); the following reaction system is adopted: cas-sf19 final concentration is 100nM, gRNA final concentration is 50nM, double stranded target nucleic acid final concentration is 5 ng/. Mu.L. Incubate at 37 ℃ for 1h. The experimental group was supplemented with gRNA and target nucleic acid, and the control group was not supplemented with gRNA.
As shown in fig. 5, cas-sf19 in the experimental group was able to cleave double-stranded nucleic acid in the system, showing a distinct cleavage band, compared to the control without gRNA. This indicates that Cas-sf19 can be used for cleavage and editing of double stranded target nucleic acids. In fig. 5, 1 is an experimental group and 2 is a control group.
While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.
Sequence listing
<110> Shunheng Biotech Co., ltd
<120> CRISPR enzymes and systems and uses
<130> P2022-0237
<150> CN202110162094.2
<151> 2021-02-05
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 911
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> Cas-sf19
<400> 1
Met Pro Ser His Lys Ser Ser Arg Val Leu Leu His Asp Val Pro Pro
1 5 10 15
Glu Leu Val Thr His Tyr Glu Ala Ser His Arg Val Ala Arg Phe Leu
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Met Glu Leu Leu Leu Ala Met Arg Gln Thr Pro Tyr Val Arg Arg Glu
35 40 45
Thr Asn Gly Glu Leu His Glu Val Thr Pro Asp Glu Ile Asp Glu Phe
50 55 60
Leu Arg Arg Tyr Thr Gly Glu Arg Leu Glu Ala Val Arg Pro Leu Leu
65 70 75 80
Lys Ser Phe Ala Glu Ala Val Leu His Glu Asp Asp Lys Glu Asn Arg
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Ala Phe Ala Lys Pro Glu Asn Ala Ala Leu Leu Leu Cys Asn Ser Glu
100 105 110
Thr Glu Ser Gly Thr Gln Tyr Phe Lys Lys Pro Gly Tyr Gln Leu Leu
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Lys Gln Ala Ile Glu Lys Lys Trp Pro Trp Lys Arg Leu Lys Gln Glu
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Leu Val Asp Glu Lys Gly Asn Ile Thr Lys Lys Phe Ala Ala Leu Ser
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Ile Glu Glu Trp Arg Asp Phe Phe Glu Ser Glu Asp Leu Asp Ala Leu
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Gly Lys Glu Leu Leu Arg Arg Thr Gln Ala Glu Gly Met Arg Ala Gly
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Arg Arg Leu Arg Glu Glu Gly Val Phe Pro Val Arg Leu Pro Asp Glu
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Leu Asp Ile Arg Ser Ser Lys Ala Ala Leu Ala Ser Val Ser Glu Arg
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Leu Lys Ser Trp Ile Asp Cys Asn Arg Arg Ala Ala Glu Gln Lys Ala
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Glu Arg Lys Gln Arg Phe Glu Arg Leu Arg Asp Ala Leu Glu Ser Ser
245 250 255
Arg Tyr Asp Leu Phe Lys Lys Phe Ala Thr Asp Leu Gln Glu Ile Asp
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Tyr Ser Val Thr Ala Arg Leu Val Gln Ala Leu Arg His Phe Pro Lys
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Arg Gln Pro Pro Glu Leu Gln Pro Ala Leu Ala Val Leu Lys Leu Asp
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Lys Tyr Arg Pro Leu Trp Glu Asn Cys Gly Glu Leu Gly Arg Thr Tyr
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Leu Ala Glu Gln Arg Trp Lys Ser His Ser Gly Arg Ala Ala Val Ser
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Phe Cys Asp Pro Asp Arg Ser Pro Ile Lys Val Arg Phe Gly Leu Thr
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Gly Arg Gly Arg Pro Phe Leu Leu Ser Ala Glu Gly Gly Arg Phe Phe
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Val Thr Leu Lys Leu Ala Cys Gly Asp Ile Gly Leu Arg Ala Leu Pro
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Ser Arg Tyr Phe Trp Asn Pro Lys Val Thr Ala His Arg Lys Asn Gly
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Lys Ser Glu Phe Asn Val Glu Phe Thr Lys Cys Thr Thr Glu Asn Arg
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Arg Phe Ala Ala His Val Lys Glu Leu Ser Ile Val Arg His Lys Gln
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Glu Thr Phe Gln Met Leu Phe Ala Val Lys Asn Leu Arg Ser Val Leu
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Gly Arg Asp Pro Asn Glu Leu Lys Ser Tyr Ile Asp Tyr Tyr Lys Asn
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Leu Arg Leu Asp Thr Leu Lys Lys Leu Thr Cys Ala Ile Val Arg Thr
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Gln Thr Ala Cys Ile Thr Asp Glu Phe Gly Tyr Arg Pro Val Arg Gly
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<210> 2
<211> 2736
<212> DNA
<213> Artificial sequence (artificial sequence)
<220>
<223> Cas-sf19
<400> 2
atgccaagcc acaaatcgag tcgtgtactt ctacacgatg tgcccccaga gctcgtaacg 60
cactacgagg catcccatcg agtcgctcgt ttcctgatgg agcttctcct cgcgatgcgg 120
cagacgccat acgtgcgccg cgagaccaac ggcgagctgc acgaagtaac accggatgag 180
atcgacgagt tcctgcggcg ttacaccggc gagcgactgg aggcagttcg cccccttctg 240
aaatccttcg ccgaggcggt gttgcacgag gacgacaaag agaatcgagc gttcgctaaa 300
cctgagaacg cggcacttct tctgtgtaac tccgaaacag agagcggcac gcaatacttc 360
aagaagcctg gatatcaatt gctcaagcag gcaatcgaga agaagtggcc atggaagcgt 420
cttaagcaag agctcgtcga cgagaaaggt aacatcacaa agaagttcgc tgcgcttagc 480
attgaggaat ggcgcgactt cttcgaatct gaagacctcg acgctctggg caaagaactc 540
ctacggcgaa cgcaggccga agggatgcga gccggccgcc gactgcgcga ggagggggtc 600
tttcccgtcc gattgcccga tgaattggac atacgcagtt cgaaagccgc attggcgtcg 660
gtttccgaac gactgaagtc ttggatcgac tgcaaccgtc gggccgcaga gcaaaaggct 720
gagcgtaaac agcgtttcga gcgactccga gacgccctcg agtcgtcgcg ctacgacctg 780
ttcaagaagt tcgcgaccga cttacaggaa atcgactaca gcgtgacggc aagattggtc 840
caagcgttga ggcattttcc gaagcgccag ccgccggagc tccaacccgc gctcgctgtt 900
ctcaaactcg ataagtaccg accactatgg gagaattgtg gcgaactcgg aaggacgtac 960
ctcgccgagc aacggtggaa gagtcactcc ggtcgcgcgg cggtttcttt ttgcgatccc 1020
gatcgcagtc cgatcaaggt ccgtttcggg ctcacagggc gggggcgacc gttccttcta 1080
tcagccgaag gcggaaggtt tttcgtcacc ctcaaactag cctgcggtga tattggcctt 1140
cgcgcccttc cgagtcgcta tttctggaat ccaaaggtga cagcgcaccg taaaaacggc 1200
aagtcggagt tcaatgtgga attcacgaaa tgcacaaccg agaatcgccg atttgccgcg 1260
cacgtcaaag agctgtccat cgttcggcac aaacagcgct actactgttt cgtcgattat 1320
ggtttcgaac ctgtgccgat atccgaggcg gcgaacaccg cgtgcaactt ttttcgggct 1380
cctctgacac agtcgcaacc gaagcccaaa gagcccgtca gtgttatggg aatcgacttg 1440
ggcgtgaatc cggcttttgc ttacgccgtc tgcacactcg gacagcagaa ggcgaaccaa 1500
atcaccgtcc ccgtggcaaa gatgaaggac acaagtttcc atgctacagg cgtgggaggt 1560
ggggtgcatg accgcaagct ccacgccgat ttgaaggaac ttgccgacac gtgtttttac 1620
ggctcgaagt acatcggtct cagtaagcgg cttcgcgacc gcgggaccct caacgagctg 1680
cagcgcaaga tccttgaaga gagatacata cccggtttca atattgtcca cgtggaggac 1740
gatcatgacc agcggcgccg caacatcggc gcgcgagtgc gtgaattgaa gagcgagttc 1800
aaacgattga ggcacgtgtt ctacgaacga cagcacgggc gtcgccgaag gccggcgccg 1860
ctgatatgtt ccgagacgtt tcagatgttg tttgccgtga agaatctccg cagcgtgctc 1920
aaggcgtgga atcggtacca ctggactagg ggcgatggcg agagccgcgg ccgtgatccg 1980
aacgaactta agtcctatat cgattactac aaaaaccttc gcctcgatac actcaagaaa 2040
cttacgtgtg cgatcgttcg gaccgcgcga tctcacggcg ttgaaatcgt ggcgttggaa 2100
gacatcaagc gagtggacta cgacgatcag gtcaagcgag ccaaggagaa cagcttgttg 2160
agtctgtggg cgccgggcat gattctggaa cgaatcgaac aggaactcgc aaacgaagga 2220
attcgcacgt ggagaatcga ccctcgacat acgagtcaaa cagcgtgcat taccgacgaa 2280
ttcgggtatc gaccagtgcg aggcaaggaa aacctattct ttgaagcgaa tggcgaattg 2340
ctgcgagtca actcggacgt gaatgcggcg atcaatattg cccgacgatt cctgacccga 2400
tatcggaaac taacgcagct ttgggctcag cctcttgaag atggagacta cctgatatct 2460
gtgaagaggc agttcgaggc ggcctttttg atggcggaaa cccgccaacc ggctgccgtg 2520
ttggtgccag agggcgaggg tatttatcga ctgaagggga tatcggggga gcgcgaaacg 2580
gagttgcggg agcaattaat gcggcgccgt tcggagaagt tctaccggca cggggagcac 2640
tggttcacgg tgaagaggca ccgcaaggcc atcgacacat tgcgcgatca agtattggag 2700
cgtggtgccc gcttgatccg cgagattcct acttaa 2736
<210> 3
<211> 36
<212> RNA
<213> Artificial sequence (artificial sequence)
<220>
<223> DR
<400> 3
gugcugccgg ucucuaaucg gggaucggaa uugcac 36

Claims (15)

1.Cas protein, characterized in that the amino acid sequence of the Cas protein is shown as SEQ ID No. 1.
2. A fusion protein comprising the Cas protein of claim 1 and other modifying moieties.
3. An isolated polynucleotide, wherein the polynucleotide is a polynucleotide sequence encoding a Cas protein of claim 1, or a polynucleotide sequence encoding a fusion protein of claim 2.
4. A vector comprising the polynucleotide of claim 3 operably linked to a regulatory element.
5. A composition, characterized in that the composition comprises:
(i) A protein component selected from: a Cas protein according to claim 1 or a fusion protein according to claim 2;
(ii) A nucleic acid component selected from the group consisting of: a gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA of a gRNA, or a precursor RNA nucleic acid encoding the gRNA, the gRNA comprising a direct repeat sequence capable of binding the Cas protein of claim 1 and a guide sequence capable of targeting a target sequence;
the protein component and the nucleic acid component are combined with each other to form a complex.
6. An activated CRISPR complex comprising:
(i) A protein component selected from: a Cas protein according to claim 1 or a fusion protein according to claim 2;
(ii) A nucleic acid component selected from the group consisting of: a gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA of a gRNA, or a precursor RNA nucleic acid encoding the gRNA, the gRNA comprising a direct repeat sequence capable of binding the Cas protein of claim 1 and a guide sequence capable of targeting a target sequence;
(iii) A target sequence bound to said gRNA.
7. An engineered host cell, wherein said host cell is of a non-animal plant variety, comprising a Cas protein of claim 1, or a fusion protein of claim 2, or a polynucleotide of claim 3, or a vector of claim 4, or a composition of claim 5, or an activated CRISPR complex of claim 6.
8. Use of the composition of claim 5, or the activated CRISPR complex of claim 6, or the host cell of claim 7 in gene editing, gene targeting or gene cleavage, for non-disease diagnostic and therapeutic purposes; alternatively, use in the manufacture of a reagent or kit for gene editing, gene targeting or gene cleavage.
9. Use of the composition of claim 5, or the activated CRISPR complex of claim 6, or the host cell of claim 7 for a use selected from any one or any of the following, for non-disease diagnostic and therapeutic purposes:
editing the target nucleic acid; cleaving double-stranded DNA, single-stranded DNA, or single-stranded RNA; detecting nucleic acid; base-editing double-stranded nucleic acids; base editing single-stranded nucleic acids.
10. A method of editing a target nucleic acid, cleaving a target nucleic acid, for non-disease diagnostic and therapeutic purposes, comprising contacting a target nucleic acid with the composition of claim 5, or the activated CRISPR complex of claim 6, or the host cell of claim 7.
11. A method of cleaving single-stranded nucleic acids, the method comprising, contacting a population of nucleic acids with the Cas protein of claim 1 and a gRNA, wherein the population of nucleic acids comprises a target nucleic acid and at least one non-target single-stranded nucleic acid, the gRNA comprising a direct repeat sequence capable of binding the Cas protein of claim 1 and a guide sequence capable of targeting a target sequence, the gRNA being capable of targeting the target nucleic acid, the Cas protein cleaving the non-target single-stranded nucleic acid.
12. A kit for gene editing, gene targeting or gene cleavage comprising the composition of claim 5, or the activated CRISPR complex of claim 6, or the host cell of claim 7.
13. A kit for detecting a target nucleic acid in a sample, the kit comprising: (a) The Cas protein of claim 1, or a nucleic acid encoding the Cas protein; (b) A gRNA, or a nucleic acid encoding the gRNA, or a precursor RNA of the gRNA, or a nucleic acid encoding the precursor RNA, the gRNA comprising a direct repeat sequence capable of binding the Cas protein of claim 1 and a guide sequence capable of targeting a target sequence; and (c) a single-stranded nucleic acid detector that is single-stranded and does not hybridize to the gRNA.
14. Use of the composition of claim 5, or the activated CRISPR complex of claim 6, or the host cell of claim 7, in the preparation of a formulation or kit for:
(i) Gene or genome editing;
(ii) Target nucleic acid detection and/or diagnosis;
(iii) Editing a target sequence in a target locus to modify an organism;
(iv) Treatment of diseases;
(v) Targeting a target gene;
(vi) Cutting the target gene.
15. A method of detecting a target nucleic acid in a sample, the method being for non-disease diagnostic and therapeutic purposes, the method comprising contacting the sample with a Cas protein of claim 1, a gRNA comprising a region that binds to the Cas protein and a guide sequence that hybridizes to the target nucleic acid, and a single-stranded nucleic acid detector; detecting a detectable signal generated by the Cas protein-cleaved single-stranded nucleic acid detector, thereby detecting a target nucleic acid; the single-stranded nucleic acid detector does not hybridize to the gRNA.
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