WO2019206233A1

WO2019206233A1 - Rna-edited crispr/cas effector protein and system

Info

Publication number: WO2019206233A1
Application number: PCT/CN2019/084340
Authority: WO
Inventors: 赖锦盛; 张湘博; 周英思; 朱金洁; 吕梦璐; 赵海铭; 宋伟彬
Original assignee: 中国农业大学
Priority date: 2018-04-25
Filing date: 2019-04-25
Publication date: 2019-10-31
Also published as: CN112020560B; CN112020560A

Abstract

The invention relates to the field of nucleic acid editing, in particular to the field of clustered regularly interspaced short palindromic repeats (CRISPR) technology. In particular, the invention relates to a Cas effector protein, a fusion protein comprising the Cas effector protein, and nucleic acid molecules encoding the same. The invention also relates to a compound and a composition for nucleic acid editing (e.g., gene or genome editing) comprising the protein or the fusion protein of the invention, or nucleic acid molecules encoding the same. The invention also relates to a method for nucleic acid editing (e.g., gene or genome editing) using the protein or the fusion protein of the invention.

Description

RNA-edited CRISPR/Cas effector protein and system

Technical field

The invention relates to the field of nucleic acid editing, in particular to the field of regularly clustered short palindrome repetition (CRISPR) technology. In particular, the invention relates to Cas effector proteins, fusion proteins comprising such proteins, and nucleic acid molecules encoding the same. The invention also relates to complexes and compositions for nucleic acid editing (e.g., gene or genome editing) comprising a protein or fusion protein of the invention, or a nucleic acid molecule encoding the same. The invention also relates to methods for nucleic acid editing (eg, gene or genome editing) using a protein or fusion protein comprising the invention.

Background technique

The CRISPR-cas system is widely found in bacteria and archaea and is adaptive to the virus. CRISPR-cas-mediated immunity mainly includes three stages: (1) adaptive stage, cas1-cas2 protein complex inserts the target DNA fragment into the CRISPR repeat region; (2) expression and processing stage, CRISPR sequence transcription and cas The effector protein processes the mature crRNA; (3) the interference phase, the cas protein and the crRNA assemble into a complex to process the target DNA or RNA.

Currently, the CRISPR-cas system has evolved into a variety of defense mechanisms, including RNA-mediated DNA or RNA editing mechanisms. The CRISPR-cas system is divided into class1 and class2. Class1 is a complex composed of multiple proteins and is relatively complex. The Class 2 system is relatively simple in structure and has only one effector protein, so it is widely used. In class 2, Type II and typeV edit DNA; Type VI CRISPR-Cas system has cleavage activity on the target RNA. Compared with the DNA-edited CRISPR-Cas system, the RNA-edited CRISPR-Cas system can regulate genes at the level of gene transcription, as well as detect live viruses, RNA interference, gene-selective splicing, and fluorescence in situ hybridization. Therefore, it is especially important to mine new RNA editing systems.

Summary of the invention

The inventors of the present application have unexpectedly discovered a novel RNA-directed ribonuclease endonuclease after extensive experimentation and repeated exploration. Based on this finding, the inventors have developed a new RNA editing CRISPR/Cas system and a gene editing method based on the system.

Cas effector protein

Accordingly, in a first aspect, the invention provides a protein having the amino acid sequence set forth in any one of SEQ ID NOs: 1-7, or an ortholog, homolog, variant or functional fragment thereof; Wherein the ortholog, homologue, variant or functional fragment substantially retains the biological function of the sequence from which it is derived.

In the present invention, the biological functions include, but are not limited to, activity binding to a targeting RNA, endoribonuclease activity, binding to a specific site of a target sequence under the guidance of a targeting RNA, and cleavage.

In certain embodiments, the ortholog, homolog, variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least compared to the sequence from which it is derived. 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In certain embodiments, the ortholog, homolog, variant has at least 80%, at least 85%, at least 90%, compared to the sequence set forth in any one of SEQ ID NOs: 1-7, 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 origin of the sequence The biological function of the sequence (eg, activity binding to the targeting RNA, endoribonuclease activity, activity that binds to a specific site of the target sequence and cleaves under the guidance of a targeting RNA).

In certain embodiments, the protein is an effector protein in the CRISPR/Cas system.

In certain embodiments, the protein of the invention comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:

(i) the sequence of any one of SEQ ID NOs: 1-7;

(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in any one of SEQ ID NOs: 1-7 (eg, 1, 2, 3, 4, 5, 6 a sequence of 7, 7 or 9 amino acid substitutions, deletions or additions; or

(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of the sequence set forth in any one of SEQ ID NOs: 1-7 a sequence of at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

(i) the sequence shown as SEQ ID NO: 1;

(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 1 (

eg

1, 2, 3, 4, 5, 6, 7) a sequence of 8, 9 or 10 amino acid substitutions, deletions or additions; or

(iii) having 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%, of the sequence set forth in SEQ ID NO: At least 97%, at least 98%, or at least 99% of sequences of sequence identity.

In certain embodiments, the protein of the invention has the amino acid sequence set forth in SEQ ID NO:1.

(i) the sequence shown as SEQ ID NO: 2;

(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 2 (eg, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 9 or 10 amino acid substitutions, deletions or additions; or

In certain embodiments, the protein of the invention has the amino acid sequence set forth in SEQ ID NO:2.

(i) the sequence shown as SEQ ID NO:3;

(ii) having one or more amino acid substitutions, deletions or additions compared to the sequence set forth in SEQ ID NO: 3 (

eg

(iii) having 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%, and the sequence set forth in SEQ ID NO: At least 97%, at least 98%, or at least 99% of sequences of sequence identity.

In certain embodiments, the protein of the invention has the amino acid sequence set forth in SEQ ID NO:3.

(i) the sequence shown as SEQ ID NO:4;

(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 4 (eg, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 9 or 10 amino acid substitutions, deletions or additions; or

In certain embodiments, the protein of the invention has the amino acid sequence set forth in SEQ ID NO:4.

(i) the sequence shown as SEQ ID NO:5;

(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 5 (eg, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 9 or 10 amino acid substitutions, deletions or additions; or

In certain embodiments, the protein of the invention has the amino acid sequence set forth in SEQ ID NO:5.

(i) the sequence shown as SEQ ID NO:6;

(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 6 (

eg

In certain embodiments, the protein of the invention has the amino acid sequence set forth in SEQ ID NO: 6.

(i) the sequence shown as SEQ ID NO:7;

(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 7 (

eg

In certain embodiments, the protein of the invention has the amino acid sequence set forth in SEQ ID NO:7.

Derived protein

A protein of the invention can be derivatized, for example, linked to another molecule (e.g., another polypeptide or protein). In general, derivatization (eg, labeling) of a protein does not affect the desired activity of the protein (eg, activity associated with targeting RNA, endonuclease activity, binding to a specific site of the target sequence under the guidance of a targeting RNA, and cleavage active). Thus, the proteins of the invention are also intended to include such derivatized forms. For example, a protein of the invention can be functionally linked (by chemical coupling, gene fusion, non-covalent attachment or otherwise) to one or more other molecular groups, such as another protein or polypeptide, a detection reagent, a pharmaceutical reagent Wait.

In particular, the proteins of the invention may be linked to other functional units. For example, it can be ligated to a nuclear localization signal (NLS) sequence to increase the ability of the proteins of the invention to enter the nucleus. For example, it can be linked to a targeting moiety to render the protein of the invention targeted. For example, it can be linked to a detectable label to facilitate detection of the proteins of the invention. For example, it can be linked to an epitope tag to facilitate expression, detection, tracing, and/or purification of the proteins of the invention.

Conjugate

Thus, in a second aspect, the invention provides a conjugate comprising a protein and a modified moiety as described above.

In certain embodiments, the modified moiety is selected from another protein or polypeptide, a detectable label, or any combination thereof.

And X. (eg, a KRAB domain or a SID domain), a nuclease domain (eg, Fok1), having a domain selected from the group consisting of methylase activity, demethylase, transcriptional activation activity, transcriptional repression Activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-stranded RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity and nucleic acid binding activity; and any combination thereof.

In certain embodiments, a conjugate of the invention comprises one or more NLS sequences, such as the NLS of the SV40 viral large T antigen. In certain exemplary embodiments, the NLS sequence is set forth in SEQ ID NO:22. In certain embodiments, the NLS sequence is located at, near, or near the end of the protein of the invention (eg, the N-terminus or the C-terminus). In certain exemplary embodiments, the NLS sequence is located at, near, or near the C-terminus of the protein of the invention.

In certain embodiments, the conjugates of the invention comprise an epitope tag. Such epitope tags are well known to those skilled in the art, examples of which include, but are not limited to, His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art know how to achieve the desired purpose (eg, Purify, test or trace) Select the appropriate epitope tag.

In certain embodiments, a conjugate of the invention comprises a reporter gene sequence. Such reporter genes are well known to those skilled in the art, and examples include, but are not limited to, GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, and the like.

In certain embodiments, the conjugates of the invention comprise a domain capable of binding to a DNA molecule or an intracellular molecule, such as a maltose binding protein (MBP), a DNA binding domain of Lex A (DBD), a DBD of GAL4, and the like. .

In certain embodiments, the conjugates of the invention comprise a detectable label, such as a fluorescent dye, such as FITC or DAPI.

In certain embodiments, a protein of the invention is optionally coupled, conjugated or fused to the modified moiety by a linker.

In certain embodiments, the modified moiety is directly linked to the N-terminus or C-terminus of the protein of the invention.

In certain embodiments, the modified moiety is linked to the N-terminus or C-terminus of the protein of the invention by a linker. Such linkers are well known in the art, examples of which include, but are not limited to, one or more (eg, 1, 2, 3, 4 or 5) amino acids (eg, Glu or Ser) or amino acid derivatives. A linker (eg, Ahx, β-Ala, GABA, or Ava), or PEG, and the like.

Fusion protein

In a third aspect, the invention provides a fusion protein comprising a protein of the invention and an additional protein or polypeptide.

In certain embodiments, a fusion protein of the invention comprises one or more NLS sequences, such as the NLS of the SV40 viral large T antigen. In certain embodiments, the NLS sequence is at, near, or near the end of the protein of the invention (eg, the N-terminus or the C-terminus). In certain exemplary embodiments, the NLS sequence is located at, near, or near the C-terminus of the protein of the invention.

In certain embodiments, a fusion protein of the invention comprises an epitope tag.

In certain embodiments, a fusion protein of the invention comprises a reporter gene sequence.

In certain embodiments, a fusion protein of the invention comprises a domain capable of binding to a DNA molecule or an intracellular molecule.

In certain embodiments, a protein of the invention is optionally fused to the additional protein or polypeptide via a linker.

In certain embodiments, the additional protein or polypeptide is directly linked to the N-terminus or C-terminus of the protein of the invention.

In certain embodiments, the additional protein or polypeptide is linked to the N-terminus or C-terminus of the protein of the invention by a linker.

In certain exemplary embodiments, the fusion proteins of the invention have an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-29.

The protein of the present invention, the conjugate of the present invention or the fusion protein of the present invention is not limited by the manner in which it is produced, for example, it can be produced by a genetic engineering method (recombination technique) or can be produced by a chemical synthesis method.

Co-directional repeat

In a fourth aspect, the invention provides an isolated nucleic acid molecule comprising a sequence selected from the group consisting of: or consisting of:

(i) the sequence set forth in any one of SEQ ID NOs: 15-21.

(ii) having one or more base substitutions, deletions or additions (for example, 1, 2, 3, 4, 5, compared to the sequence shown in any one of SEQ ID NOs: 15-21) a sequence of 6, 7, 8, or 10 base substitutions, deletions, or additions;

(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the sequence set forth in any one of SEQ ID NOs: 15-21. a sequence of at least 95% sequence identity;

(iv) a sequence that hybridizes under stringent conditions to the sequence set forth in any one of (i)-(iii); or

(v) the complement of the sequence set forth in any of (i)-(iii);

Furthermore, the sequence of any one of (ii)-(v) substantially retains the biological function of the sequence from which it is derived, the biological function of the sequence being referred to as the same direction in the CRISPR-Cas system Repeat the activity of the sequence.

In certain embodiments, the isolated nucleic acid molecule is a direct repeat in a CRISPR-Cas system.

In certain embodiments, the isolated nucleic acid molecule comprises one or more stem loops or an optimized secondary structure. In certain embodiments, the sequence of any of (ii)-(v) retains the secondary structure of the sequence from which it is derived.

In certain embodiments, the nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:

(a) the nucleotide sequence shown in any one of SEQ ID NOs: 15-21.

(b) a sequence that hybridizes under stringent conditions to the sequence described in (a); or

(c) the complement of the sequence described in (a).

In certain embodiments, the isolated nucleic acid molecule is RNA.

In certain embodiments, the isolated nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of:

(i) the sequence of SEQ ID NO: 15;

(ii) a substitution, deletion or addition of one or more bases compared to the sequence shown in SEQ ID NO: 15 (

eg

1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO: Sequence of sequence identity;

(v) the complement of the sequence set forth in any of (i)-(iii).

(a) the nucleotide sequence shown in SEQ ID NO: 15;

(c) the complement of the nucleotide sequence shown by SEQ ID NO: 15.

(i) the sequence shown as SEQ ID NO:16;

(ii) having one or more base substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 16 (for example, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(v) the complement of the sequence set forth in any of (i)-(iii).

(a) the nucleotide sequence shown in SEQ ID NO: 16;

(c) the complement of the nucleotide sequence shown by SEQ ID NO: 16.

(i) the sequence shown as SEQ ID NO:17;

(ii) having one or more base substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 17 (for example, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(v) the complement of the sequence set forth in any of (i)-(iii).

(a) the nucleotide sequence shown in SEQ ID NO: 17;

(b) a sequence that hybridizes under stringent conditions to the sequence described in (a);

(c) the complement of the nucleotide sequence shown by SEQ ID NO: 17.

(i) the sequence shown as SEQ ID NO:18;

(ii) a substitution, deletion or addition of one or more bases compared to the sequence shown in SEQ ID NO: 18 (

eg

(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO: 18. Sequence of sequence identity;

(v) the complement of the sequence set forth in any of (i)-(iii).

(a) the nucleotide sequence shown in SEQ ID NO: 18;

(c) the complement of the nucleotide sequence shown by SEQ ID NO: 18.

(i) the sequence shown as SEQ ID NO:19;

(ii) having one or more base substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 19 (for example, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(v) the complement of the sequence set forth in any of (i)-(iii).

(a) the nucleotide sequence shown in SEQ ID NO: 19;

(c) the complement of the nucleotide sequence shown by SEQ ID NO: 19.

(i) the sequence shown as SEQ ID NO:20;

(ii) having one or more base substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 20 (for example, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(v) the complement of the sequence set forth in any of (i)-(iii).

(a) the nucleotide sequence shown in SEQ ID NO:20;

(c) the complement of the nucleotide sequence shown by SEQ ID NO: 20.

(i) the sequence shown as SEQ ID NO:21.

(ii) a substitution, deletion or addition of one or more bases compared to the sequence shown in SEQ ID NO: 21 (for example, 1, 2, 3, 4, 5, 6, 7 a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:21. Sequence of sequence identity;

(v) the complement of the sequence set forth in any of (i)-(iii).

(a) the nucleotide sequence shown as SEQ ID NO: 21;

(c) the complement of the nucleotide sequence shown by SEQ ID NO:21.

CRISPR/Cas Complex

In a fifth aspect, the invention provides a composite comprising:

(i) a protein component selected from the group consisting of a protein, conjugate or fusion protein of the invention, and any combination thereof;

(ii) a nucleic acid component comprising an isolated nucleic acid molecule as described above and a targeting sequence capable of hybridizing to the target sequence,

Wherein the protein component and the nucleic acid component are combined with each other to form a complex.

In certain embodiments, the targeting sequence is linked to the 3' or 5' end of the nucleic acid molecule.

In certain embodiments, the targeting sequence comprises the complement of the target sequence.

In certain embodiments, the nucleic acid component is a targeting RNA in a CRISPR/Cas system.

In certain embodiments, the nucleic acid molecule is RNA.

In certain embodiments, the complex does not comprise a trans-acting tracrRNA.

Encoding nucleic acids, vectors and host cells

In a sixth aspect, the invention provides an isolated nucleic acid molecule comprising:

(i) a nucleotide sequence encoding a protein or fusion protein of the invention;

(ii) an isolated nucleic acid molecule encoding a fourth aspect; or

(iii) a nucleotide sequence comprising (i) and (ii).

In certain embodiments, the nucleotide sequence set forth in any of (i)-(iii) is codon optimized for expression in a prokaryotic cell. In certain embodiments, the nucleotide sequence set forth in any of (i)-(iii) is codon optimized for expression in eukaryotic cells.

In a seventh aspect, the invention provides a vector comprising the isolated nucleic acid molecule of the sixth aspect. The vector of the present invention may be a cloning vector or an expression vector. In certain embodiments, vectors of the invention are, for example, plasmids, cosmids, phage, cosmid, and the like. In certain selected embodiments, the vector is capable of expressing a protein of the invention, a fusion protein, an isolated nucleic acid molecule of the fourth aspect, or a fifth aspect, in a subject (eg, a mammal, eg, a human) Said complex.

In an eighth aspect, the invention also provides a host cell comprising an isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, such as mouse cells, human cells, etc.). The cells of the invention may also be cell lines, such as 293T cells. In certain embodiments, the host cell is a prokaryotic cell.

Composition and carrier composition

In a ninth aspect, the present invention also provides a composition comprising:

(i) a first component selected from the group consisting of a protein, a conjugate, a fusion protein, a nucleotide sequence encoding the protein or fusion protein, and any combination thereof;

(ii) a second component which is a nucleotide sequence comprising a targeting RNA or a nucleotide sequence encoding the nucleotide sequence comprising the targeting RNA;

Wherein the targeting RNA comprises a homologous repeat sequence and a targeting sequence, the targeting sequence being capable of hybridizing to the target sequence;

The targeting RNA is capable of forming a complex with the protein, conjugate or fusion protein described in (i).

In certain embodiments, the isotropic repeat is an isolated nucleic acid molecule as defined in the fourth aspect.

In certain embodiments, the targeting sequence is ligated to the 3' or 5' end of the homologous repeat. In certain embodiments, the targeting sequence comprises the complement of the target sequence.

In certain embodiments, the composition does not comprise tracrRNA.

In certain embodiments, the composition is non-naturally occurring or modified. In certain embodiments, at least one component of the composition is non-naturally occurring or modified. In certain embodiments, the first component is non-naturally occurring or modified; and/or the second component is non-naturally occurring or modified.

In certain embodiments, the target sequence is an RNA sequence from a prokaryotic or eukaryotic cell. In certain embodiments, the target sequence is a non-naturally occurring RNA sequence.

In certain embodiments, the target sequence is present in a cell. In certain embodiments, the target sequence is present within the nucleus or within the cytoplasm (eg, an organelle). In certain embodiments, the cell is a prokaryotic cell. In certain embodiments, the cell is a eukaryotic cell.

In certain embodiments, the protein is linked to one or more NLS sequences. In certain embodiments, the conjugate or fusion protein comprises one or more NLS sequences. In certain embodiments, the NLS sequence is linked to the N-terminus or C-terminus of the protein. In certain embodiments, the NLS sequence is fused to the N-terminus or C-terminus of the protein.

In a tenth aspect, the present invention also provides a composition comprising one or more carriers, the one or more carriers comprising:

(i) a first nucleic acid which is a nucleotide sequence encoding a protein or fusion protein of the invention; optionally the first nucleic acid is operably linked to a first regulatory element;

(ii) a second nucleic acid encoding a nucleotide sequence comprising a targeting RNA; optionally the second nucleic acid is operably linked to a second regulatory element;

among them:

The first nucleic acid and the second nucleic acid are present on the same or different carrier;

The targeting RNA comprises a homologous repeat sequence and a targeting sequence capable of hybridizing to the target sequence;

The targeting RNA is capable of forming a complex with the effector protein or fusion protein described in (i).

In certain embodiments, the composition does not comprise tracrRNA.

In certain embodiments, the composition is non-naturally occurring or modified. In certain embodiments, at least one component of the composition is non-naturally occurring or modified.

In certain embodiments, the first regulatory element is a promoter, such as an inducible promoter.

In certain embodiments, the second regulatory element is a promoter, such as an inducible promoter.

In certain embodiments, one type of vector is a plasmid, which refers to a circular double stranded DNA loop in which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector in which a virus-derived DNA or RNA sequence is present for packaging a virus (eg, retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated In the vector of the virus). The viral vector also comprises a polynucleotide carried by a virus for transfection into a host cell. Certain vectors (e.g., bacterial vectors having bacterial origins of replication and episomal mammalian vectors) are capable of autonomous replication in the host cell into which they are introduced. Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell and thereby 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." Common expression vectors used in recombinant DNA techniques are typically in the form of plasmids.

The recombinant expression vector can comprise a nucleic acid molecule of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector comprises one or more regulatory elements selected based on the host cell to be used for expression. The regulatory element is operably linked to the nucleic acid sequence to be expressed.

Delivery and delivery composition

The protein, the conjugate, the fusion protein of the invention, the isolated nucleic acid molecule of the fourth aspect, the complex of the invention, the isolated nucleic acid molecule of the sixth aspect, the vector of the seventh aspect The compositions of the ninth and tenth aspects 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, cation transfection, lipofection, dendritic Transfection, heat shock transfection, nuclear transfection, magnetic transfection, lipofection, puncture transfection, optical transfection, reagent-enhanced nucleic acid uptake, and via liposomes, immunoliposomes, viral particles, artificial viruses Delivery of body, etc.

Accordingly, in another aspect, the present invention provides a delivery composition comprising a delivery vehicle, and one or more selected from the group consisting of a protein, a conjugate, a fusion protein of the invention, as in the fourth aspect The isolated nucleic acid molecule, the complex of the invention, the isolated nucleic acid molecule of the sixth aspect, the carrier of the seventh aspect, the composition of the ninth and tenth aspects.

In certain embodiments, the delivery vehicle is a particle.

In certain embodiments, the delivery vehicle is selected from the group consisting of 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 (eg, replication defective reverse transcription) Virus, lentivirus, adenovirus or adeno-associated virus).

Kit

In another aspect, the invention provides a kit comprising one or more of the components described above. In certain embodiments, the kit comprises one or more components selected from the group consisting of a protein, a conjugate, a fusion protein of the invention, an isolated nucleic acid molecule of the fourth aspect, the invention The complex, the isolated nucleic acid molecule of the sixth aspect, the carrier of the seventh aspect, the composition of the ninth and tenth aspects.

In certain embodiments, the kit of the invention comprises the composition of the ninth aspect. In certain embodiments, the kit further comprises instructions for using the composition.

In certain embodiments, the kit of the invention comprises the composition of the tenth aspect. In certain embodiments, the kit further comprises instructions for using the composition.

In certain embodiments, the components contained in the kits of the invention can be provided in any suitable container.

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

In certain embodiments, the kit further comprises one or more oligonucleotides corresponding to a targeting sequence for insertion into a vector for operably linking the guide Sequence and adjustment elements.

In certain embodiments, the kit further comprises an RNA template. In certain embodiments, the RNA template comprises an RNA sequence encoding a protein or a non-coding RNA sequence (eg, a microRNA).

Method and use

In another aspect, the invention provides a method of modifying a target sequence, comprising: the complex of the fifth aspect, the composition of the ninth aspect, the composition of the tenth aspect Or the delivery composition as described herein is contacted with the target sequence or delivered to a cell comprising the target sequence; wherein the target sequence is associated with or is present in the gene of interest And the target sequence is RNA.

In certain embodiments, the target sequence is present in a cell. 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 selected from a non-human primate, bovine, porcine or rodent cell. In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a poultry or fish. In certain embodiments, the cell is a plant cell, such as a cell of a cultivated plant (such as cassava, corn, sorghum, wheat, or rice), algae, tree, or vegetable.

In certain embodiments, the target sequence is present in a nucleic acid molecule (eg, a plasmid) in vitro. In certain embodiments, the target sequence is present in a plasmid.

In certain embodiments, the modification refers to a cleavage of the target sequence, such as a double strand break or a single strand break.

In certain embodiments, the target sequence is a ssRNA.

In certain embodiments, the method further comprises contacting the RNA template with the target sequence or delivering to a cell comprising the target sequence. In certain embodiments, the modification further comprises inserting an RNA template (eg, an exogenous nucleic acid) into the fragment.

In certain embodiments, the protein, conjugate, fusion protein, isolated nucleic acid molecule, complex, vector or composition is included in a delivery vehicle.

In certain embodiments, the delivery vehicle is selected from the group consisting of a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a viral vector (eg, a replication defective retrovirus, a lentivirus, an adenovirus) Or adeno-associated virus).

In certain embodiments, the methods are for RNA interference or modulation of gene expression.

In certain embodiments, the methods modulate gene expression by modulating RNA processing or RNA activation (RNAa). In such embodiments, the RNA processing can include RNA splicing (including alternative splicing), viral replication (eg, satellite viruses, phage or retroviruses, eg, HBV, HCV, HIV, etc.) or tRNA biosynthesis. In some cases, the RNAa promotes gene expression. Thus, in such embodiments, the method inhibits gene expression by interfering with or reducing RNAa.

In another aspect, the invention relates to the protein of the first aspect, the conjugate of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid molecule of the fourth aspect, The complex of claim 5, the isolated nucleic acid molecule of the sixth aspect, the carrier of the seventh aspect, the composition of the ninth aspect, the composition of the tenth aspect A kit or delivery composition of the invention, for use in one or more of the following, or in the preparation of a formulation for one or more selected from the group consisting of:

(1) modifying the target sequence;

(2) RNA interference; or

(3) Regulate gene expression.

In another aspect, the invention provides a method of detecting a target sequence, comprising the complex of the fifth aspect, the composition of the ninth aspect, the composition of the tenth aspect or A delivery composition as described herein is contacted with the target sequence or delivered to a cell comprising the target sequence; wherein the target sequence is RNA.

In certain embodiments, a targeting sequence contained in the complex, composition or delivery composition is capable of hybridizing to the target sequence.

In certain embodiments, the target sequence is present in a nucleic acid molecule in vitro.

In certain embodiments, the target sequence is present in a cell. In certain embodiments, the cell is a prokaryotic cell.

In certain embodiments, the cell is a living cell.

In certain embodiments, the protein component comprised by the complex, composition or delivery composition carries a detectable label.

In certain embodiments, the protein component comprised by the complex, composition or delivery composition is fused with a fluorescent protein (eg, GFP).

In certain embodiments, the method is northern blotting. In such embodiments, the northern blotting method involves isolating RNA samples by size by electrophoresis. Thus, the complexes or compositions of the invention can be used to specifically bind and detect target RNA sequences.

In certain embodiments, the method is fluorescence in situ hybridization. In such embodiments, the protein component of the complex, composition or delivery composition of the invention carries a detectable label (eg, a fluorescent label).

In another aspect, the invention relates to the protein of the first aspect, the conjugate of the second aspect, the fusion protein of the third aspect, the isolated nucleic acid molecule of the fourth aspect, The complex of claim 5, the isolated nucleic acid molecule of the sixth aspect, the carrier of the seventh aspect, the composition of the ninth aspect, the composition of the tenth aspect A kit or delivery composition of the invention for use in detecting a target sequence, or in the preparation of a preparation for detecting a target sequence.

In certain embodiments, when the CRISPR/Cas complex of the invention binds to a target RNA, Cas13e in the complex is activated, followed by cleavage of any nearby ssRNA sequence (ie, collateral cleavage) ). Once Cas13e is primed by the target RNA, other (non-complementary) RNA molecules can be cleaved. This confounding RNA cleavage can cause cytotoxicity or otherwise affect cell physiology or cellular status.

In a further aspect, the invention provides a method of modulating the state of a target cell, comprising the composition of the fifth aspect, the composition of the ninth aspect, as described in the tenth aspect The composition or delivery composition as described herein is introduced into the target cell.

In certain embodiments, a target sequence that hybridizes to a targeting sequence contained in the complex, composition, or delivery composition is present in the target cell.

In certain embodiments, the modulating the state of the target cell comprises:

(1) inducing cell dormancy in vitro or in vivo;

(2) Inducing cell cycle arrest in vitro or in vivo;

(3) inhibiting cell growth and/or cell proliferation in vitro or in vivo;

(4) inducing cell anergy in vitro or in vivo;

(5) inducing apoptosis in vitro or in vivo;

(6) inducing cell necrosis in vitro or in vivo;

(7) inducing cell death in vitro or in vivo; or

(8) Inducing programmed cell death in vitro or in vivo.

In certain embodiments, the cell is a prokaryotic cell.

(1) inducing cell dormancy in vitro or in vivo;

(2) Inducing cell cycle arrest in vitro or in vivo;

(3) inhibiting cell growth and/or cell proliferation in vitro or in vivo;

(4) inducing cell anergy in vitro or in vivo;

(5) inducing apoptosis in vitro or in vivo;

(6) inducing cell necrosis in vitro or in vivo;

(7) inducing cell death in vitro or in vivo; or

(8) Inducing programmed cell death in vitro or in vivo.

In the present invention, the methods as described above may be therapeutic or prophylactic, and may target a particular target cell, cell (sub)pop, or cell/tissue type. In certain instances, the particular cell, cell (sub)pop, or cell/tissue type expresses one or more target sequences, such as one or more specific target RNAs. In certain embodiments, non-limiting examples of such target cells include tumor cells that express a particular transcript, a given class of neurons, cells that cause autoimmunity, or cells that are infected with a particular pathogen (eg, a virus).

Accordingly, in certain embodiments, the present invention also provides a method of treating a pathological condition characterized by the presence of a defective cell, comprising administering to a subject in need thereof a complex as described in the fifth aspect The composition of the ninth aspect, the composition of the tenth aspect, or the delivery composition as described herein. In certain embodiments, the invention relates to the protein of the first aspect, the conjugate of the second aspect, the fusion protein of the third aspect, the isolated of the fourth aspect The nucleic acid molecule, the complex according to the fifth aspect, the isolated nucleic acid molecule according to the sixth aspect, the carrier according to the seventh aspect, the composition according to the ninth aspect, according to the tenth aspect Use of the composition, kit of the invention or delivery composition for the treatment of a pathological condition characterized by the presence of defective cells. It will be appreciated that in the above embodiments, the complex or composition of the invention preferably targets a specific target sequence of the defective cell.

In certain embodiments, the pathological condition characterized by the presence of a defective cell is a tumor. Accordingly, in certain embodiments, the present invention also provides a method of treating a tumor comprising administering to a subject in need thereof a complex according to the fifth aspect, as described in the ninth aspect A composition, a composition as described in the tenth aspect, or a delivery composition as described herein. In certain embodiments, the invention relates to the protein of the first aspect, the conjugate of the second aspect, the fusion protein of the third aspect, the isolated of the fourth aspect The nucleic acid molecule, the complex according to the fifth aspect, the isolated nucleic acid molecule according to the sixth aspect, the carrier according to the seventh aspect, the composition according to the ninth aspect, according to the tenth aspect Use of the composition, kit of the invention or delivery composition for the treatment of a tumor. In such embodiments, the complex or composition of the invention preferably targets a tumor cell specific target sequence.

In certain embodiments, the pathological condition characterized by the presence of a defective cell is a disease caused by a pathogen infection or infection by a pathogen. Accordingly, in certain embodiments, the present invention also provides a method of treating a pathogen infection or a disease caused by a pathogen infection, comprising administering to a subject in need thereof a compound as described in the fifth aspect The composition of the ninth aspect, the composition of the tenth aspect, or the delivery composition as described herein. In certain embodiments, the invention relates to the protein of the first aspect, the conjugate of the second aspect, the fusion protein of the third aspect, the isolated of the fourth aspect The nucleic acid molecule, the complex according to the fifth aspect, the isolated nucleic acid molecule according to the sixth aspect, the carrier according to the seventh aspect, the composition according to the ninth aspect, according to the tenth aspect Use of a composition, a kit of the invention or a delivery composition for treating a pathogen infection or a disease caused by a pathogen infection. In such embodiments, the complex or composition of the invention preferably targets a specific target sequence (e.g., a target sequence derived from the pathogen) of a cell infected by the pathogen.

In certain embodiments, the pathological condition characterized by the presence of a defective cell is an autoimmune disease. Accordingly, in certain embodiments, the present invention also provides a method of treating an autoimmune disease, comprising administering to a subject in need thereof a complex according to the fifth aspect, as in the ninth aspect A composition, a composition as described in the tenth aspect, or a delivery composition as described herein. In certain embodiments, the invention relates to the protein of the first aspect, the conjugate of the second aspect, the fusion protein of the third aspect, the isolated of the fourth aspect The nucleic acid molecule, the complex according to the fifth aspect, the isolated nucleic acid molecule according to the sixth aspect, the carrier according to the seventh aspect, the composition according to the ninth aspect, according to the tenth aspect Use of the composition, kit of the invention or delivery composition for the treatment of an autoimmune disease. In such embodiments, the complex or composition of the invention preferably targets a specific target sequence of a cell (eg, a particular immune cell) that causes the autoimmune disease.

In certain embodiments, the invention relates to the protein of the first aspect, the conjugate of the second aspect, the fusion protein of the third aspect, the isolated of the fourth aspect The nucleic acid molecule, the complex according to the fifth aspect, the isolated nucleic acid molecule according to the sixth aspect, the carrier according to the seventh aspect, the composition according to the ninth aspect, according to the tenth aspect The composition, the kit or delivery composition of the invention, the use in a method of treatment, or the use in the preparation of a formulation for use in a method of treatment. Such methods of treatment include gene editing, transcriptome editing, or gene therapy.

Cell and cell progeny

In some cases, modifications introduced to cells by the methods of the invention can cause the cells and their progeny to be altered to improve the production of their biological products, such as antibodies, starch, ethanol, or other desired cellular output. In some cases, modifications introduced into the cell by the methods of the invention can cause the cell and its progeny to include changes that result in a change in the produced biological product.

Thus, in another aspect, the invention relates to a cell or a progeny thereof obtained by the method as described above, wherein the cell contains a modification that is not found in its wild type.

In certain embodiments, the modification results in a change in transcription or translation of at least one RNA product. In certain embodiments, the modification results in increased expression of at least one RNA product. In certain embodiments, the modification results in decreased expression of at least one RNA product.

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, such as a human cell.

The invention also relates to a cell product of a cell or a progeny thereof as described above.

The invention also relates to an in vitro, ex vivo or in vivo cell or cell line or a progeny thereof, the cell or cell line or a progeny thereof comprising: the protein of the first aspect, such as the second The conjugate of the aspect, the fusion protein of the third aspect, the isolated nucleic acid molecule of the fourth aspect, the complex of the fifth aspect, the isolated nucleic acid of the sixth aspect A molecule, a carrier according to the seventh aspect, a composition according to the ninth aspect, a composition according to the tenth aspect, a kit of the invention or a delivery composition.

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, such as a cell of a non-human primate, cow, sheep, pig, dog, monkey, rabbit, rodent (eg, rat or mouse). In certain embodiments, the cell is a non-mammalian eukaryotic cell, such as a poultry bird (eg, chicken), a fish or a crustacean (eg, scorpion, shrimp) cells. In certain embodiments, the cell is a plant cell, such as a cell possessed by a monocot or a dicot or a cultivated plant or a cell of a food crop such as cassava, corn, sorghum, soybean, wheat, oat or rice, for example Algae, tree or production of plants, fruits or vegetables (for example, trees such as citrus, nut trees; nightshade, cotton, tobacco, tomatoes, grapes, coffee, cocoa, etc.).

In certain embodiments, the cell is a stem cell or stem cell line.

Definition of Terms

In the present invention, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art, unless otherwise stated. Furthermore, the procedures of molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA used herein are routine steps widely used in the corresponding fields. . Also, for a better understanding of the present invention, definitions and explanations of related terms are provided below.

In the present invention, the expression "Cas13e" refers to a Cas effector protein first discovered and identified by the present inventors having an amino acid sequence selected from the group consisting of:

(i) the sequence of any one of SEQ ID NOs: 1-7;

The Cas13e of the present invention is an endonuclease which binds to a specific site of a target RNA sequence and cleaves under the guidance of a guide RNA.

As used herein, the term "regular clustered interspersed short palindrome repeat (CRISPR)-CRISPR-related (Cas) (CRISPR-Cas) system" or "CRISPR system" is used interchangeably and has skill in the art. A person generally understands the meaning, which typically includes a transcription product or other element associated with the expression of a CRISPR-associated ("Cas") gene, or a transcription product or other element capable of directing the activity of the Cas gene. Such transcription products or other elements may comprise a sequence encoding a Cas effector protein and a targeting RNA comprising CRISPR RNA (crRNA), and a trans-acting crRNA (tracrRNA) sequence contained in the CRISPR-Cas9 system, or from a CRISPR locus Other sequences or transcripts. In the Cas13e-based CRISPR system of the present invention, tracrRNA sequences are not required.

As used herein, the terms "Cas effector protein", "Cas effector enzyme" are used interchangeably and refer to any of the proteins presented in the CRISPR-Cas system that are greater than 900 amino acids in length. In some cases, such proteins refer to proteins identified from the Cas locus.

As used herein, the terms "guide RNA", "mature crRNA" are used interchangeably and have the meaning as commonly understood by one of ordinary skill in the art. In general, the targeting RNA may comprise a direct repeat sequence and a guide sequence, or consist essentially of or consist of a homologous repeat sequence and a guide sequence (also referred to as a spacer sequence in the context of an endogenous CRISPR system). (spacer)) composition. In some cases, the targeting sequence is any polynucleotide sequence that is sufficiently complementary to the target sequence to hybridize to the target sequence and direct the specific binding of the CRISPR/Cas complex to the target sequence. In certain embodiments, when optimally aligned, the degree of complementarity between the targeting 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%. Determining the optimal alignment is within the abilities of one of ordinary skill in the art. For example, there are publicly available and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in Matlab, Bowtie, Geneious, Biopython, and SeqMan.

In some cases, the targeting sequence is at least 5, at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 in length, At least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45 or at least 50 Nucleotides. In some cases, the guide sequence is no more than 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 15 in length. , 10 or fewer nucleotides.

In some cases, the isotropic repeats are at least 10, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22 in length. , at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, At least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65 or at least 70 nucleotides . In some cases, the same direction repeat sequence is no more than 70, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56 in length. , 55, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 15 , 10 or fewer nucleotides.

As used herein, the term "CRISPR/Cas complex" refers to a ribonucleoprotein complex formed by the binding of a guide RNA or a mature crRNA to a Cas protein, which comprises hybridization to a target sequence and with Cas Protein-directed targeting sequences. The ribonucleoprotein complex is capable of recognizing and cleaving a polynucleotide that hybridizes to the targeting RNA or mature crRNA.

Thus, in the context of the formation of a CRISPR/Cas complex, a "target sequence" refers to a polynucleotide that is designed to be targeted by a targeting sequence, such as a sequence that is complementary to the targeting sequence, wherein the target Hybridization between the sequence and the targeting sequence will promote the formation of the CRISPR/Cas complex. Complete complementarity is not required as long as sufficient complementarity exists to cause hybridization and promote the formation of a CRISPR/Cas complex. In the present invention, the target sequence is RNA. Therefore, "target sequence" and "target RNA" are used interchangeably in the present invention. The target sequence can be any suitable form of RNA, such as mRNA, tRNA, rRNA, miRNA, siRNA or shRNA. In the present invention, the expression "target sequence" may be any endogenous or exogenous RNA sequence to a cell (eg, a eukaryotic cell). In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence can be located in an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. A sequence or template that can be used to integrate into an RNA sequence comprising the target sequence is referred to as an "RNA template." In certain embodiments, the RNA template is an exogenous nucleic acid.

In some cases, the target RNA can be a sequence encoding a gene product (eg, a protein) (eg, mRNA or pre-mRNA) or a non-coding sequence (eg, ncRNA, lncRNA, tRNA or rRNA). Non-limiting examples of target RNA include sequences associated with signaling biochemical pathways (eg, signaling biochemical pathway-associated RNA) or disease-associated RNA. The "disease-associated RNA" refers to an RNA sequence produced by the RNA sequence in an abnormal level or abnormal form in a tissue or cell affected by the disease, compared to a tissue or a cell not having a disease control. presence. The "disease-associated RNA" may be transcribed from a gene having an abnormally elevated expression level, or may be an RNA transcribed from a gene having abnormally decreased expression, wherein the altered expression level is related to the occurrence and/or development of the disease. . Disease-associated RNA also refers to RNA transcribed from a gene having a mutation or gene mutation that is directly responsible for or is in linkage disequilibrium with the gene causing the disease. The translated product may be known or unknown and may be at a normal or abnormal level.

In some cases, the target RNA can comprise interfering RNA (ie, RNA present in the RNA interference pathway, eg, shRNA, siRNA, etc.). In certain embodiments, the target RNA is a microRNA (miRNA).

As used herein, the term "wild type" has the meaning commonly understood by those skilled in the art to mean a typical form of a organism, a strain, a gene, or a feature that distinguishes it from a mutant or variant when it exists in nature. It can be isolated from sources in nature and not intentionally modified.

As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and refer to artificial participation. When these terms are used to describe a nucleic acid molecule or polypeptide, it is meant that the nucleic acid molecule or polypeptide is at least substantially freed from at least one other component of its association in nature or as found in nature.

As used herein, the term "orthologue, ortholog" has the meaning as commonly understood by one of ordinary skill in the art. As a further guide, an "ortholog" of a protein as referred to herein refers to a protein belonging to a different species that performs the same or similar function as a protein that is an ortholog thereof.

As used herein, the term "identity" is used to mean the matching of sequences between two polypeptides or between two nucleic acids. When a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position. The "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table. The gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences. In addition, the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package (available at www.gcg.com) can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .

The term "vector," as used herein, refers to a nucleic acid vehicle into which a polynucleotide can be inserted. A vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide. The vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC). Phage such as lambda phage or M13 phage and animal virus. Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40). 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 also contain an origin of replication.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, such as a fungal cell such as a yeast cell or an Aspergillus. For example, S2 Drosophila cells or insect cells such as Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

One of skill in the art will appreciate that the design of the expression vector can depend on factors such as the choice of host cell to be transformed, the level of expression desired, and the like. A vector can be introduced into a host cell to thereby produce a transcript, protein, or peptide, including a protein, fusion protein, isolated nucleic acid molecule, etc. as described herein (eg, a CRISPR transcript, such as a nucleic acid transcript) , protein, or enzyme).

As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (eg, transcription termination signals, such as polyadenylation signals and Poly U sequence), a detailed description can be found in Goeddel, GENE EXPRE SSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego ), California (1990). In certain instances, regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in a plurality of types of host cells, as well as those sequences that direct expression of the nucleotide sequence only in certain host cells (eg, Tissue-specific regulatory sequence). Tissue-specific promoters can primarily direct expression in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (eg, liver, pancreas), or specific cell types (eg, Lymphocytes). In some cases, the regulatory elements may also direct expression in a time-dependent manner (eg, in a cell cycle dependent or developmental stage dependent manner), which may or may not be tissue or cell type specific. In some cases, the term "regulatory element" encompasses enhancer elements such as WPRE; CMV enhancer; R-U5' fragment in LTR of HTLV-I ((Mol. Cell. Biol., 8th ( 1) Vol., pp. 466-472, 1988); SV40 enhancer; and intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981).

As used herein, the term "promoter" has the meaning well-known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of the gene that initiates expression of the downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in a gene product in the cell under most or all physiological conditions of the cell. The production. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in substantially only when an inducer corresponding to the promoter is present in the cell The gene product is produced intracellularly. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, is substantially only caused when the cell is a cell of the tissue type corresponding to the promoter Gene products are produced in the cells.

As used herein, the term "operably linked" is intended to mean that a nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows expression of the nucleotide sequence (eg, In an in vitro transcription/translation system or in the host cell when the vector is introduced into a host cell).

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 conventional Watson-Crick or other non-traditional types. Percent complement indicates the percentage of residues in a nucleic acid molecule that can form a hydrogen bond (eg, Watson-Crick base pairing) with a second nucleic acid sequence (eg, 5, 6, 7, 8 out of 10) 9, 10, that is 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Completely complementary" means that all contiguous residues of one nucleic acid sequence form a hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "substantially complementary" means having 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98 in the region of 30, 35, 40, 45, 50 or more nucleotides %, 99%, or 100% complementarity, or two nucleic acids that hybridize under stringent conditions.

As used herein, "stringent conditions" for hybridization refers to conditions under which a nucleic acid that is complementary to a target sequence primarily hybridizes to the target sequence and does not substantially hybridize to a non-target sequence. Stringent conditions are usually sequence dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in "Technology Techniques In Biochemi stry And Molecular Biology-Hybridization With Nucleic Acid Probes" by Tijssen (1993). ), Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, New York.

As used herein, the term "hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that hydrogen bonds through the bases between these nucleotide residues. And stabilized. Hydrogen bonding can occur by means of Watson-Crick base pairing, Hoogstein binding or in any other sequence specific manner. The complex may comprise two chains forming one duplex, three or more chains forming a multi-strand complex, a single self-hybridizing strand, or any combination of these. The hybridization reaction can constitute a step in a broader process, such as the initiation of PCR, or the cleavage of a polynucleotide via an enzyme. A sequence that is capable of hybridizing to a given sequence is referred to as the "complement" of the given sequence.

As used herein, the term "expression" refers to a process by which a DNA template is transcribed into a polynucleotide (eg, transcribed into mRNA or other RNA transcript) and/or transcribed mRNA, which is subsequently translated into a peptide, The process of a 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 can include splicing of mRNA in eukaryotic cells.

As used herein, the term "linker" refers to a linear polypeptide formed by the joining of multiple amino acid residues by peptide bonds. The linker of the invention may be a synthetic amino acid sequence, or a naturally occurring polypeptide sequence, such as a polypeptide having the function of a hinge region. Such linker polypeptides are well known in the art (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al. (1994) Structure 2: 1121- 1123).

As used herein, the term "treating" refers to treating or curing a condition, delaying the onset of symptoms of the condition, and/or delaying the progression of the condition.

As used herein, the term "subject" includes, but is not limited to, various animals, such as mammals, such as bovine, equine, ovine, porcine, canine, feline, A rabbit, a rodent (eg, a mouse or rat), a non-human primate (eg, a macaque or a cynomolgus monkey) or a human.

Advantageous effects of the invention

The Cas effector protein and system of the invention can efficiently perform RNA editing, detecting living virus, RNA interference, gene selective scission, fluorescence in situ hybridization, etc., and realizing regulation of genes at the level of gene transcription. This will provide important resources for new applications in genomic engineering and biotechnology.

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The various objects and advantageous aspects of the invention will be apparent to those skilled in the <

DRAWINGS

Figure 1 shows the two HEPN domains of the cas13e protein.

Figure 2 shows the secondary structure of the repeat sequence of cas13e. A, B, C, D, E, F, and G are the repeat secondary structures of cas13e1.1, cas13e1.2, cas13e1.3, cas13e1.4, cas13e1.5, cas13e2.1, and cas13e2.2, respectively.

Figure 3 shows the activity of cas13e1.3 protein on the processing of pre-crRNA in bacteria.

Figure 4 shows the in vitro processing activity of cas13e1.3 protein on pre-crRNA.

Sequence information

Information on the partial sequences involved in the present invention is provided in Table 1 below.

Table 1: Description of the sequence

SEQ ID NO:SEQ ID NO:	描述 description
11	Cas13e1.1的氨基酸序列Amino acid sequence of Cas13e1.1
22	Cas13e1.2的氨基酸序列Amino acid sequence of Cas13e1.2
33	Cas13e1.3的氨基酸序列Amino acid sequence of Cas13e1.3
44	Cas13e1.4的氨基酸序列Amino acid sequence of Cas13e1.4
55	Cas13e1.5的氨基酸序列Amino acid sequence of Cas13e1.5
66	Cas13e2.1的氨基酸序列Amino acid sequence of Cas13e2.1
77	Cas13e2.2的氨基酸序列Amino acid sequence of Cas13e2.2
88	Cas13e1.1的编码核酸序列Encoding nucleic acid sequence of Cas13e1.1
99	Cas13e1.2的编码核酸序列Encoding nucleic acid sequence of Cas13e1.2
1010	Cas13e1.3的编码核酸序列Encoding nucleic acid sequence of Cas13e1.3
1111	Cas13e1.4的编码核酸序列Encoding nucleic acid sequence of Cas13e1.4

1212	Cas13e1.5的编码核酸序列Encoding nucleic acid sequence of Cas13e1.5
1313	Cas13e2.1的编码核酸序列Encoding nucleic acid sequence of Cas13e2.1
1414	Cas13e2.2的编码核酸序列Encoding nucleic acid sequence of Cas13e2.2
1515	Cas13e1.1/原型同向重复序列Cas13e1.1/prototype repeating sequence
1616	Cas13e1.2/原型同向重复序列Cas13e1.2/prototype repeating sequence
1717	Cas13e1.3/原型同向重复序列Cas13e1.3/prototype repeating sequence
1818	Cas13e1.4/原型同向重复序列Cas13e1.4/prototype repeating sequence
1919	Cas13e1.5/原型同向重复序列Cas13e1.5/prototype repeating sequence
2020	Cas13e2.1/原型同向重复序列Cas13e2.1/prototype repeating sequence
21twenty one	Cas13e2.2/原型同向重复序列Cas13e2.2/prototype repeating sequence
22twenty two	NLS的氨基酸序列NLS amino acid sequence
23twenty three	Cas13e1.1-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas13e1.1-NLS fusion protein
24twenty four	Cas13e1.2-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas13e1.2-NLS fusion protein
2525	Cas13e1.3-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas13e1.3-NLS fusion protein
2626	Cas13e1.4-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas13e1.4-NLS fusion protein
2727	Cas13e1.5-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas13e1.5-NLS fusion protein
2828	Cas13e2.1-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas13e2.1-NLS fusion protein
2929	Cas13e2.2-NLS融合蛋白的氨基酸序列Amino acid sequence of Cas13e2.2-NLS fusion protein
3030	Cas13e1.1/CRISPR阵列Cas13e1.1/CRISPR Array
3131	Cas13e1.2/CRISPR阵列Cas13e1.2/CRISPR Array
3232	Cas13e1.3/CRISPR阵列Cas13e1.3/CRISPR Array
3333	Cas13e1.4/CRISPR阵列Cas13e1.4/CRISPR array
3434	Cas13e1.5/CRISPR阵列Cas13e1.5/CRISPR Array
3535	Cas13e2.1/CRISPR阵列Cas13e2.1/CRISPR array
3636	Cas13e2.2/CRISPR阵列Cas13e2.2/CRISPR Array

detailed description

The invention is described with reference to the following examples which are intended to illustrate, but not limit the invention.

The experiments and methods described in the Examples are carried out essentially according to conventional methods well known in the art and described in various references, unless otherwise indicated. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention can be found in Sambrook, Fry ( Fritsch) and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (FMAubel et al., (1987)); METHOS IN ENZYMOLOGY series (academic publishing company): "PCR 2: Practical Methods" (PCR 2: A PRACTICAL) APPROACH) (MJ MacPherson, BD Hames, and GR Taylor (1995)), Harlow, and Lane Editor (1988) : ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELLCULTURE (RI Freshney, eds. (1987)).

In addition, those which do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially. The invention is described by way of example, and is not intended to limit the scope of the invention. All publications and other references mentioned herein are incorporated by reference in their entirety.

Unless otherwise specified, the sequence synthesis involved in the following examples was completed by Nanjing Kingsray Biotechnology Co., Ltd., and the sequencing involved was completed by Shanghai Yingjun Biotechnology Co., Ltd.

Example 1. Identification of Cas13e protein and Cas13e targeting RNA

First download NCBI bacterial genomic data and JGI metagenomic data. Contig containing the CRISPR sequence was searched using the primercr 1.06 software, and then the protein was predicted using the MetaGeneMark software for the contig, and a protein of >=800 amino acids was screened. The function of the protein was annotated using the Hmmsearch method, excluding the cas protein of known function and the protein with significant functional domain annotation in the nr database (E-value<=e-10). The mcl software classifies the proteins retained.

On this basis, the inventors obtained and identified a novel family of Cas effector proteins, namely Cas13e, which can be divided into two subfamilies, namely cas13e.1 and cas13e.2. The family protein sequences are designated Cas13e1.1 (SEQ ID NO: 1), Cas13e1.2 (SEQ ID NO: 2), Cas13e1.3 (SEQ ID NO: 3), Cas13e1.4 (SEQ ID NO: 4), Cas13e1.5 (SEQ ID NO: 5), Cas13e2.1 (SEQ ID NO: 6), Cas13e2.2 (SEQ ID NO: 7), the DNA encoding these seven proteins are as shown in SEQ ID NOs: 8-14, respectively. Show. The prototype homologous repeat sequences corresponding to the seven proteins (prototype repeat sequence, the repeat sequence contained in the pre-crRNA) are shown in SEQ ID NOs: 15-21. The two HEPN domains of the above seven cas13e proteins were obtained by performing multiple sequence alignment of the proteins to analyze the functional domains containing "RxxxxH" (Fig. 1). Further, the secondary structure of the prototype repeat sequence of the above seven cas13e proteins was obtained by analysis by Vienna RNA software (Fig. 2).

Example 2. Processing of pre-crRNA by Cas13e protein

2.1 Cas13e1.3 protein processing activity of pre-crRNA in E. coli:

1. A double-stranded DNA molecule of SEQ ID NO: 10 is artificially synthesized, and a double-stranded DNA molecule encoding SEQ ID NO: 32 (CRISPR array: repeat + spacer 1 + repeat + spacer + repeat) is artificially synthesized.

2. The double-stranded DNA molecule synthesized in the step 1 was ligated to the prokaryotic expression vector pACYC-Duet-1 to obtain a recombinant plasmid pACYC-Duet-1-CRISPR/cas13e1.3. The recombinant plasmid pACYC-Duet-1-CRISPR/cas13e1.3 was subjected to one-generation sequencing to confirm the DNA sequence described in the step 1.

3. The recombinant plasmid pACYC-Duet-1-CRISPR/cas13e1.3 was introduced into Escherichia coli EC100 to obtain a recombinant strain, and the recombinant strain was named EC100-CRISPR/cas13e1.3.

4. The monoclonal clone of EC100-CRISPR/cas13e1.3 was taken, inoculated into 100 mL of LB liquid medium (containing 50 μg/mL ampicillin), and cultured at 37 ° C, shaking at 200 rpm for 12 hours to obtain a culture bacterial solution.

5. Extraction of bacterial RNA: Transfer 1.5 mL of the bacterial culture to a pre-chilled microcentrifuge tube, centrifuge at 6,000 x g for 5 minutes at 4 °C. After centrifugation, the supernatant was discarded, and the cell pellet was resuspended in 200 μL MaxBacterial Enhancement Reagent preheated to 95 ° C, and mixed by pipetting. Incubate at 95 ° C for 4 minutes. Add 1 mL to the lysate

Mix well by pipetting and incubate for 5 minutes at room temperature. Add 0.2 mL of cold chloroform, mix by shaking the tube for 15 seconds, and incubate for 2-3 minutes at room temperature. Centrifuge at 12,000 x g for 15 minutes at 4 °C. 600 μL of the supernatant was taken in a new tube, and 0.5 mL of cold isopropanol was added to precipitate RNA, which was mixed by inversion and incubated at room temperature for 10 minutes. Centrifuge at 15,000 x g for 10 minutes at 4 ° C, discard the supernatant, add 1 mL of 75% ethanol, and vortex to mix. Centrifuge at 7500 x g for 5 minutes at 4 ° C, discard the supernatant, and air dry. The RNA pellet was dissolved in 50 μL of RNase-free water and incubated at 60 ° C for 10 minutes.

6. Digestion of DNA: 20 ug of RNA was dissolved in 39.5 μL dH ₂ O at 65 ° C for 5 min. On ice for 5 min, 0.5 μL of RNAI, 5 μL of buffer, 5 μL of DNase I, and 37 ° C for 45 min (50 μL system) were added. Add 50 μL dH ₂ O and adjust the volume to 100 μL. After centrifugation of 2 mL Phase-Lock tube at 16000 g for 30 s, 100 μL of phenol:chloroform:isoamyl alcohol (25:24:1), 100 μL of digested RNA was added, shaken for 15 s, centrifuged at 16000 g for 12 min at 15 ° C. The supernatant was taken in a new 1.5 mL centrifuge tube, and an equal volume of isopropanol 1/10 NaoAC was added to the supernatant for 1 h or -20 ° C overnight. Centrifuge at 16000 g for 30 min at 4 ° C, discard the supernatant. The precipitate was washed by adding 350 μL of 75% ethanol, centrifuged at 16000 g for 10 min at 4 ° C, and the supernatant was discarded. Dry, add 20 μL of RNase-free water, 65 ° C, and dissolve the precipitate for 5 min. NanoDrop measures the concentration and runs the glue.

7, 3' dephosphorylation and 5' phosphorylation: ~20 μg of digested RNA, each added to 42.5 μL, 90 ° C for 2 min. Cool on ice for 5 min. 5 μL of 10×T4 PNK buffer, 0.5 μL of RNaI, 2 μL of T4PNK (50 μL), and 37 ° C for 6 h were added. Add 1 μL of T4PNK, 1.25 μL (100 mM) ATP, 1 hour at 37 °C. Add 47.75 μL dH ₂ O and adjust the volume to 100 μL. After centrifugation of 2 mL Phase-Lock tube at 16000 g for 30 s, 100 μL of phenol:chloroform:isoamyl alcohol (25:24:1), 100 μL of digested RNA was added, shaken for 15 s, centrifuged at 16000 g for 12 min at 15 ° C. The supernatant was taken in a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added to the supernatant in a total volume of 1/10 NaoAC for 1 h or -20 ° C overnight. Centrifuge at 16000 g for 30 min at 4 ° C, discard the supernatant. The precipitate was washed by adding 350 μL of 75% ethanol, centrifuged at 16000 g for 10 min at 4 ° C, and the supernatant was discarded. Dry, add 21 μL of RNase-free water, 65 ° C, dissolve the precipitate for 5 min, and measure the concentration by NanoDrop.

8. RNA monophosphorylation: 20 μL of RNA, 1 min at 90 ° C, and cooled on ice for 5 min. 2 μL of RNA 5'Polphosphatase 10×Reaction buffer, 0.5 μL of Inhibitor, 1 μL of RNA 5'Polphosphatase (20 Units), RNase-free water to 20 μL, and 37 ° C for 60 min were added. Add 80 μL dH ₂ O and adjust the volume to 100 μL. After centrifugation of 2 mL Phase-Lock tube 16000 g for 30 s, 100 μL of phenol:chloroform:isoamyl alcohol (25:24:1), 100 μL of digested RNA was added, shaken for 15 s, centrifuged at 16000 g for 12 min at 15 ° C. The supernatant was taken in a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added to the supernatant in a total volume of 1/10 NaoAC for 1 h or -20 ° C overnight. After centrifugation at 16000 g for 30 min at 4 ° C, the supernatant was discarded, and the precipitate was washed by adding 350 μL of 75% ethanol, centrifuged at 16000 g for 10 min at 4 ° C, and the supernatant was discarded. Dry, add 21 μL of RNase-free water, 65 ° C, dissolve the precipitate for 5 min, and measure the concentration by NanoDrop.

9. Preparation of cDNA library: 16.5 μL RNase-free water. 5 μL Poly(A) Polymerase 10×Reaction buffer. 5 μL of 10 mM ATP. 1.5 μL RiboGuard RNase Inhibitor. 20 μL RNA Substrate. 2 μL Poly(A) Polymerase (4 Units). 50 μL total volume. 37 ° C for 20 min. Add 50 μL dH ₂ O and adjust the volume to 100 μL. After centrifugation of 2 mL Phase-Lock tube 16000 g for 30 s, 100 μL of phenol:chloroform:isoamyl alcohol (25:24:1), 100 μL of digested RNA was added, shaken for 15 s, centrifuged at 16000 g for 12 min at 15 ° C. The supernatant was taken in a new 1.5 mL centrifuge tube, and an equal volume of isopropanol was added to the supernatant in a total volume of 1/10 NaoAC for 1 h or -20 ° C overnight. After centrifugation at 16000 g for 30 min at 4 ° C, the supernatant was discarded, air-dried, 11 μL of RNase-free water was added, and the precipitate was dissolved at 65 ° C for 5 min, and the concentration was measured by NanoDrop.

10. The cDNA library was added to the sequencing linker and sent to Beijing Berry Hekang for sequencing.

11. Perform mass filtering on the raw data to remove sequences with a base average mass value below 30. After removing the linker from the sequence, the RNA sequence of 25 nt to 50 nt was retained and aligned to the reference sequence of the CRISPR array using bowtie. The results are shown in Fig. 3. The peak shape is the structure of the second-generation sequencing sequence alignment of the CRISPR block, the vertical line shows the enzyme cleavage site, the gray rectangle is the Repeat structure diagram, and the dark gray diamond shape is the spacer sequence structure diagram. Through the cleavage site information obtained by Cas13e1.3 alignment, it can be known that the pre-crRNA of Cas13e1.3 can be successfully processed into mature crRNA by Cas13e1.3 in E. coli, which consists of 27 nt Repeat sequence and 32 nt spacer. Sequence construction; structural prediction and visual analysis of mature crRNA using ViennaRNA and VARNA showed that the 3' end of the Repeat sequence of the crRNA (mature Repeat sequence) can form a 18-base-sized neck ring.

2.2 In vitro processing activity of Cas13e1.3 protein on pre-crRNA:

1. Transcription of pre-crRNA:

Primers for designing a precrRNA transcript template. The structure of the transcription template is: T7 promoter + CRISPR array of Cas13e1.3 (SEQ ID NO: 32). The primers were designed using Primer 5.0 software to ensure that the upstream and downstream primers have overlapping sequences of at least 18 bp. A double stranded DNA template was obtained using a primer annealing procedure. The template was purified using a MinElute PCR Purifcation Kit, and the concentration was measured with Nanodrop, and stored at -20 ° C until use. RNA transcription was performed using NEB's HiScribe T7 high-efficiency RNA synthesis kit, and the PCR reaction procedure was set to: 37 ° C / 3 h or 31 ° C / forever, DNAseI was added, 37 ° C / 45 min.

2. Purification of precrRNA:

Use the mixture phenol: chloroform: isoamyl alcohol (25:24:1) to remove DNAseI from the system; run the gel and purify the pre-crRNA from the polyacrylamide gel, using ZR Small-RNATM PAGE Recovery from ZYMO RESEARCH The kit was purified and recovered for pre-crRNA, and the concentration was measured with Nanodrop, and stored at -80 ° C for use.

3. Establish an in vitro enzyme digestion system

(1) Configure the following reaction system, gently pipette and mix briefly and then centrifuge briefly. Placed at 37 ° C, 1 h;

RNA Cleavage Buffer (10 * may be configured liquor): 40mM Tris-HCl (pH = 8.0), 50mM KCl, 0.5mM TECP, 10mM MgCl 2.

In addition, in order to verify whether Cas13e1.3 is an EDTA-sensitive protein, an EDTA group was also designed, which added EDTA to the reaction system.

(2) To the above reaction system, 10 μl of 2× RNA loading dye was added and placed at 98 ° C for 3 min. Immediately after the end of the reaction, it was placed on ice for 2 min;

(3) 10% TBE-Urea polyacrylamide gel loading well 10μl, 150V, 40min;

(4) The SYBR-Gold nucleic acid gel dye was added to 1×TBE running buffer, placed in a gel, and stained for 10 minutes at room temperature.

The electrophoresis results are shown in Figure 4. Cas13e1.3 is capable of cleaving pre-crRNA in vitro and is not an EDTA-sensitive protein.

While the invention has been described in detail, it will be understood by the . The invention is generally divided into the appended claims and any equivalents thereof.

Claims

A protein having the amino acid sequence set forth in any one of SEQ ID NOs: 1-7, or an ortholog, homolog, variant or functional fragment thereof; wherein the ortholog a homologue, variant or functional fragment substantially retains the biological function of the sequence from which it is derived;

For example, the ortholog, homolog, variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94 compared to the sequence from which it is derived. %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

For example, the ortholog, homolog, variant has at least 80%, at least 85%, at least 90%, at least 91%, at least compared to the sequence set forth in any one of SEQ ID NOs: 1-7. 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 the sequence from which it is derived ;

For example, the protein is an effector protein in the CRISPR/Cas system.
The protein of claim 1 comprising a sequence selected from the group consisting of: or consisting of a sequence selected from the group consisting of:

(i) the sequence of any one of SEQ ID NOs: 1-7;

(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in any one of SEQ ID NOs: 1-7 (eg, 1, 2, 3, 4, 5, 6 a sequence of 7, 7 or 9 amino acid substitutions, deletions or additions; or

(iii) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of the sequence set forth in any one of SEQ ID NOs: 1-7 a sequence of at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

For example, the protein has the amino acid sequence set forth in any one of SEQ ID NOs: 1-7.
The protein according to claim 1 or 2, which comprises a sequence selected from the following or consists of a sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO: 1;

(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 1 (eg 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 9 or 10 amino acid substitutions, deletions or additions; or

(iii) having 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%, of the sequence set forth in SEQ ID NO: a sequence of at least 97%, at least 98%, or at least 99% sequence identity;

For example, the protein has the amino acid sequence set forth in SEQ ID NO: 1.
The protein according to claim 1 or 2, which comprises a sequence selected from the following or consists of a sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO: 2;

(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 2 (eg, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 9 or 10 amino acid substitutions, deletions or additions; or

(iii) having 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%, of the sequence set forth in SEQ ID NO: a sequence of at least 97%, at least 98%, or at least 99% sequence identity;

For example, the protein has the amino acid sequence set forth in SEQ ID NO: 2.
The protein according to claim 1 or 2, which comprises a sequence selected from the following or consists of a sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO:3;

(ii) having one or more amino acid substitutions, deletions or additions compared to the sequence set forth in SEQ ID NO: 3 (eg 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 9 or 10 amino acid substitutions, deletions or additions; or

(iii) having 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%, and the sequence set forth in SEQ ID NO: a sequence of at least 97%, at least 98%, or at least 99% sequence identity;

For example, the protein has the amino acid sequence set forth in SEQ ID NO:3.
The protein according to claim 1 or 2, which comprises a sequence selected from the following or consists of a sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO:4;

(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 4 (eg, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 9 or 10 amino acid substitutions, deletions or additions; or

(iii) having 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%, of the sequence set forth in SEQ ID NO: a sequence of at least 97%, at least 98%, or at least 99% sequence identity;

For example, the protein has the amino acid sequence set forth in SEQ ID NO:4.
The protein according to claim 1 or 2, which comprises a sequence selected from the following or consists of a sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO:5;

(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 5 (eg, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 9 or 10 amino acid substitutions, deletions or additions; or

(iii) having 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%, and the sequence set forth in SEQ ID NO: a sequence of at least 97%, at least 98%, or at least 99% sequence identity;

For example, the protein has the amino acid sequence set forth in SEQ ID NO:5.
The protein according to claim 1 or 2, which comprises a sequence selected from the following or consists of a sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO:6;

(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 6 (eg 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 9 or 10 amino acid substitutions, deletions or additions; or

(iii) having 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%, of the sequence set forth in SEQ ID NO: a sequence of at least 97%, at least 98%, or at least 99% sequence identity;

For example, the protein has the amino acid sequence set forth in SEQ ID NO: 6.
The protein according to claim 1 or 2, which comprises a sequence selected from the following or consists of a sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO:7;

(ii) a substitution, deletion or addition of one or more amino acids compared to the sequence set forth in SEQ ID NO: 7 (eg 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 9 or 10 amino acid substitutions, deletions or additions; or

(iii) having 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%, and the sequence set forth in SEQ ID NO: a sequence of at least 97%, at least 98%, or at least 99% sequence identity;

For example, the protein has the amino acid sequence set forth in SEQ ID NO: 7.
A conjugate comprising the protein of any one of claims 1-9 and a modified moiety;

For example, the modified moiety is selected from additional proteins or polypeptides, detectable labels, and any combination thereof;

For example, the modified moiety is optionally linked to the N-terminus or C-terminus of the protein via a linker;

For example, the modified moiety is fused to the N-terminus or C-terminus of the protein;

For example, the additional protein or polypeptide is selected from the group consisting of an epitope tag, a reporter gene sequence, a nuclear localization signal (NLS) sequence, a targeting moiety, a transcriptional activation domain (eg, VP64), a transcriptional repression domain (eg, a KRAB structure) a domain or SID domain), a nuclease domain (eg, Fok1), having a domain selected from the group consisting of methylase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release factor Activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-stranded RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity and nucleic acid binding activity; and any combination thereof;

For example, the conjugate comprises an epitope tag;

For example, the conjugate comprises an NLS sequence;

For example, the NLS sequence is set forth in SEQ ID NO:22;

For example, the NLS sequence is located at, near, or near the end of the protein (eg, the N-terminus or the C-terminus).
A fusion protein comprising the protein of any one of claims 1-9 and an additional protein or polypeptide;

For example, the additional protein or polypeptide is optionally linked to the N-terminus or C-terminus of the protein via a linker;

For example, the additional protein or polypeptide is selected from the group consisting of an epitope tag, a reporter gene sequence, a nuclear localization signal (NLS) sequence, a targeting moiety, a transcriptional activation domain (eg, VP64), a transcriptional repression domain (eg, a KRAB structure) a domain or SID domain), a nuclease domain (eg, Fok1), having a domain selected from the group consisting of methylase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release factor Activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-stranded RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity and nucleic acid binding activity; and any combination thereof;

For example, the fusion protein comprises an epitope tag;

For example, the fusion protein comprises an NLS sequence;

For example, the NLS sequence is set forth in SEQ ID NO:22;

For example, the NLS sequence is located at, near, or near the end of the protein (eg, the N-terminus or the C-terminus);

For example, the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-29.
An isolated nucleic acid molecule comprising a sequence selected from the group consisting of: or consisting of a sequence selected from the group consisting of:

(i) the sequence set forth in any one of SEQ ID NOs: 15-21.

(ii) having one or more base substitutions, deletions or additions (for example, 1, 2, 3, 4, 5, compared to the sequence shown in any one of SEQ ID NOs: 15-21) a sequence of 6, 7, 8, or 10 base substitutions, deletions, or additions;

(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the sequence set forth in any one of SEQ ID NOs: 15-21. a sequence of at least 95% sequence identity;

(iv) a sequence that hybridizes under stringent conditions to the sequence set forth in any one of (i)-(iii); or

(v) the complement of the sequence set forth in any of (i)-(iii);

Furthermore, the sequence of any one of (ii)-(v) substantially retains the biological function of the sequence from which it is derived;

For example, the nucleic acid molecule comprises one or more stem loops or an optimized secondary structure;

For example, the sequence of any of (ii)-(v) retains the secondary structure of the sequence from which it is derived;

For example, the nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:

(a) the nucleotide sequence shown in any one of SEQ ID NOs: 15-21.

(b) a sequence that hybridizes under stringent conditions to the sequence described in (a); or

(c) the complement of the sequence described in (a);

For example, the isolated nucleic acid molecule is RNA;

For example, the isolated nucleic acid molecule is a homologous repeat in the CRISPR/Cas system.
The isolated nucleic acid molecule of claim 12 comprising a sequence selected from the group consisting of: or consisting of a sequence selected from the group consisting of:

(i) the sequence of SEQ ID NO: 15;

(ii) a substitution, deletion or addition of one or more bases compared to the sequence shown in SEQ ID NO: 15 (eg 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO: Sequence of sequence identity;

(iv) a sequence that hybridizes under stringent conditions to the sequence set forth in any one of (i)-(iii); or

(v) the complement of the sequence set forth in any of (i)-(iii);

For example, the nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:

(a) the nucleotide sequence shown in SEQ ID NO: 15;

(b) a sequence that hybridizes under stringent conditions to the sequence described in (a); or

(c) the complement of the nucleotide sequence shown by SEQ ID NO: 15.
The isolated nucleic acid molecule of claim 12 comprising a sequence selected from the group consisting of: or consisting of a sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO:16;

(ii) having one or more base substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 16 (for example, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO: Sequence of sequence identity;

(iv) a sequence that hybridizes under stringent conditions to the sequence set forth in any one of (i)-(iii); or

(v) the complement of the sequence set forth in any of (i)-(iii);

For example, the nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:

(a) the nucleotide sequence shown in SEQ ID NO: 16;

(b) a sequence that hybridizes under stringent conditions to the sequence described in (a); or

(c) the complement of the nucleotide sequence shown by SEQ ID NO: 16.
The isolated nucleic acid molecule of claim 12 comprising a sequence selected from the group consisting of: or consisting of a sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO:17;

(ii) having one or more base substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 17 (for example, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO: Sequence of sequence identity;

(iv) a sequence that hybridizes under stringent conditions to the sequence set forth in any one of (i)-(iii); or

(v) the complement of the sequence set forth in any of (i)-(iii);

For example, the nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:

(a) the nucleotide sequence shown in SEQ ID NO: 17;

(b) a sequence that hybridizes under stringent conditions to the sequence described in (a);

(c) the complement of the nucleotide sequence shown by SEQ ID NO: 17.
The isolated nucleic acid molecule of claim 12 comprising a sequence selected from the group consisting of: or consisting of a sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO:18;

(ii) a substitution, deletion or addition of one or more bases compared to the sequence shown in SEQ ID NO: 18 (eg 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO: 18. Sequence of sequence identity;

(iv) a sequence that hybridizes under stringent conditions to the sequence set forth in any one of (i)-(iii); or

(v) the complement of the sequence set forth in any of (i)-(iii);

For example, the nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:

(a) the nucleotide sequence shown in SEQ ID NO: 18;

(b) a sequence that hybridizes under stringent conditions to the sequence described in (a);

(c) the complement of the nucleotide sequence shown by SEQ ID NO: 18.
The isolated nucleic acid molecule of claim 12 comprising a sequence selected from the group consisting of: or consisting of a sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO:19;

(ii) having one or more base substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 19 (for example, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO: Sequence of sequence identity;

(iv) a sequence that hybridizes under stringent conditions to the sequence set forth in any one of (i)-(iii); or

(v) the complement of the sequence set forth in any of (i)-(iii);

For example, the nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:

(a) the nucleotide sequence shown in SEQ ID NO: 19;

(b) a sequence that hybridizes under stringent conditions to the sequence described in (a);

(c) the complement of the nucleotide sequence shown by SEQ ID NO: 19.
The isolated nucleic acid molecule of claim 12 comprising a sequence selected from the group consisting of: or consisting of a sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO:20;

(ii) having one or more base substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 20 (for example, 1, 2, 3, 4, 5, 6, 7) a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO: Sequence of sequence identity;

(iv) a sequence that hybridizes under stringent conditions to the sequence set forth in any one of (i)-(iii); or

(v) the complement of the sequence set forth in any of (i)-(iii);

For example, the nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:

(a) the nucleotide sequence shown in SEQ ID NO:20;

(b) a sequence that hybridizes under stringent conditions to the sequence described in (a);

(c) the complement of the nucleotide sequence shown by SEQ ID NO: 20.
The isolated nucleic acid molecule of claim 12 comprising a sequence selected from the group consisting of: or consisting of a sequence selected from the group consisting of:

(i) the sequence shown as SEQ ID NO:21.

(ii) a substitution, deletion or addition of one or more bases compared to the sequence shown in SEQ ID NO: 21 (for example, 1, 2, 3, 4, 5, 6, 7 a sequence of 8, 8 or 10 base substitutions, deletions or additions;

(iii) having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the sequence set forth in SEQ ID NO:21. Sequence of sequence identity;

(iv) a sequence that hybridizes under stringent conditions to the sequence set forth in any one of (i)-(iii); or

(v) the complement of the sequence set forth in any of (i)-(iii);

For example, the nucleic acid molecule comprises a sequence selected from the group consisting of: or consists of a sequence selected from the group consisting of:

(a) the nucleotide sequence shown as SEQ ID NO: 21;

(b) a sequence that hybridizes under stringent conditions to the sequence described in (a);

(c) the complement of the nucleotide sequence shown by SEQ ID NO:21.
A composite comprising:

(i) a protein component selected from the group consisting of: the protein of any one of claims 1-9, the conjugate of claim 10, the fusion protein of claim 11, and any combination thereof;

(ii) a nucleic acid component comprising the isolated nucleic acid molecule of any one of claims 12-19 and a targeting sequence capable of hybridizing to the target sequence,

Wherein the protein component and the nucleic acid component are combined with each other to form a complex;

For example, the targeting sequence is ligated to the 3' or 5' end of the nucleic acid molecule;

For example, the targeting sequence comprises the complement of the target sequence;

For example, the nucleic acid component is a targeting RNA in a CRISPR/Cas system;

For example, the nucleic acid molecule is RNA;

For example, the complex does not comprise tracrRNA.
The composite of claim 20 comprising:

(i) a protein component selected from the group consisting of the protein of claim 3, a conjugate comprising the protein, or a fusion protein;

(ii) a nucleic acid component comprising the isolated nucleic acid molecule of claim 13 and the targeting sequence.
The composite of claim 20 comprising:

(i) a protein component selected from the group consisting of the protein of claim 4, a conjugate comprising the protein, or a fusion protein;

(ii) a nucleic acid component comprising the isolated nucleic acid molecule of claim 14 and the targeting sequence.
The composite of claim 20 comprising:

(i) a protein component selected from the group consisting of the protein of claim 5, a conjugate comprising the protein, or a fusion protein;

(ii) a nucleic acid component comprising the isolated nucleic acid molecule of claim 15 and the targeting sequence.
The composite of claim 20 comprising:

(i) a protein component selected from the group consisting of the protein of claim 6, a conjugate comprising the protein, or a fusion protein;

(ii) a nucleic acid component comprising the isolated nucleic acid molecule of claim 16 and the targeting sequence.
The composite of claim 20 comprising:

(i) a protein component selected from the group consisting of the protein of claim 7, a conjugate comprising the protein, or a fusion protein;

(ii) a nucleic acid component comprising the isolated nucleic acid molecule of claim 17 and the targeting sequence.
The composite of claim 20 comprising:

(i) a protein component selected from the group consisting of the protein of claim 8, a conjugate comprising the protein, or a fusion protein;

(ii) a nucleic acid component comprising the isolated nucleic acid molecule of claim 18 and the targeting sequence.
The composite of claim 20 comprising:

(i) a protein component selected from the group consisting of the protein of claim 9, a conjugate comprising the protein, or a fusion protein;

(ii) a nucleic acid component comprising the isolated nucleic acid molecule of claim 19 and the targeting sequence.
An isolated nucleic acid molecule comprising:

(i) a nucleotide sequence encoding the protein of any one of claims 1-9, or the fusion protein of claim 11;

(ii) a nucleotide sequence encoding the isolated nucleic acid molecule of any one of claims 12-19; and/or,

(iii) a nucleotide sequence comprising (i) and (ii);

For example, the nucleotide sequence set forth in any of (i)-(iii) is codon optimized for expression in prokaryotic or eukaryotic cells.
A vector comprising the isolated nucleic acid molecule of claim 28.
A host cell comprising the isolated nucleic acid molecule of claim 28 or the vector of claim 29.
A composition comprising:

(i) a first component selected from the group consisting of the protein of any one of claims 1-9, the conjugate of claim 10, the fusion protein of claim 11, encoding the protein or fusion a nucleotide sequence of a protein, and any combination thereof; and

(ii) a second component which is a nucleotide sequence comprising a targeting RNA or a nucleotide sequence encoding the nucleotide sequence comprising the targeting RNA;

Wherein the targeting RNA comprises a homologous repeat sequence and a targeting sequence, the targeting sequence being capable of hybridizing to the target sequence;

The targeting RNA is capable of forming a complex with the protein, conjugate or fusion protein described in (i);

For example, the isotropic repeat is an isolated nucleic acid molecule as defined in any one of claims 12-19;

For example, the targeting sequence is ligated to the 3' or 5' end of the homologous repeat;

For example, the targeting sequence comprises the complement of the target sequence;

For example, the composition does not comprise tracrRNA;

For example, the composition is non-naturally occurring or modified;

For example, at least one component of the composition is non-naturally occurring or modified;

For example, the first component is non-naturally occurring or modified; and/or the second component is non-naturally occurring or modified.
The composition of claim 31 wherein:

The first component is selected from the group consisting of: the protein of claim 3, or a conjugate or fusion protein comprising the protein, or a nucleotide sequence encoding the protein or fusion protein, and any combination thereof;

The isotropic repeat sequence is an isolated nucleic acid molecule as defined in claim 13.
The composition of claim 31 wherein:

The first component is selected from the group consisting of: the protein of claim 4, or a conjugate or fusion protein comprising the protein, or a nucleotide sequence encoding the protein or fusion protein, and any combination thereof;

The isotropic repeat is an isolated nucleic acid molecule as defined in claim 14.
The composition of claim 31 wherein:

The first component is selected from the group consisting of: the protein of claim 5, or a conjugate or fusion protein comprising the protein, or a nucleotide sequence encoding the protein or fusion protein, and any combination thereof;

The isotropic repeat is an isolated nucleic acid molecule as defined in claim 15.
The composition of claim 31 wherein:

The first component is selected from the group consisting of: the protein of claim 6, or a conjugate or fusion protein comprising the protein, or a nucleotide sequence encoding the protein or fusion protein, and any combination thereof;

The isotropic repeat is an isolated nucleic acid molecule as defined in claim 16.
The composition of claim 31 wherein:

The first component is selected from the group consisting of: the protein of claim 7, or a conjugate or fusion protein comprising the protein, or a nucleotide sequence encoding the protein or fusion protein, and any combination thereof;

The isotropic repeat is an isolated nucleic acid molecule as defined in claim 17.
The composition of claim 31 wherein:

The first component is selected from the group consisting of: the protein of claim 8, or a conjugate or fusion protein comprising the protein, or a nucleotide sequence encoding the protein or fusion protein, and any combination thereof;

The isotropic repeat is an isolated nucleic acid molecule as defined in claim 18.
The composition of claim 31 wherein:

The first component is selected from the group consisting of: the protein of claim 9, or a conjugate or fusion protein comprising the protein, or a nucleotide sequence encoding the protein or fusion protein, and any combination thereof;

The isotropic repeat sequence is an isolated nucleic acid molecule as defined in claim 19.
A composition comprising one or more carriers, the one or more carriers comprising:

(i) a first nucleic acid, which is a nucleotide sequence encoding the protein of any one of claims 1-9 or the fusion protein of claim 11; optionally the first nucleic acid is operably linked to First adjustment element;

(ii) a second nucleic acid encoding a nucleotide sequence comprising a targeting RNA; optionally the second nucleic acid is operably linked to a second regulatory element;

among them:

The first nucleic acid and the second nucleic acid are present on the same or different carrier;

The targeting RNA comprises a homologous repeat sequence and a targeting sequence capable of hybridizing to the target sequence;

The targeting RNA is capable of forming a complex with the effector protein or fusion protein described in (i);

For example, the isotropic repeat is an isolated nucleic acid molecule as defined in any one of claims 12-19;

For example, the targeting sequence is ligated to the 3' or 5' end of the homologous repeat;

For example, the targeting sequence comprises the complement of the target sequence;

For example, the composition does not comprise tracrRNA;

For example, the composition is non-naturally occurring or modified;

For example, at least one component of the composition is non-naturally occurring or modified;

For example, the first regulatory element is a promoter, such as an inducible promoter;

For example, the second regulatory element is a promoter, such as an inducible promoter.
The composition of claim 39 wherein:

The first nucleic acid is a nucleotide sequence encoding the protein of claim 3 or a fusion protein comprising the protein;

The isotropic repeat sequence is an isolated nucleic acid molecule as defined in claim 13.
The composition of claim 39 wherein:

The first nucleic acid is a nucleotide sequence encoding the protein of claim 4 or a fusion protein comprising the protein;

The isotropic repeat is an isolated nucleic acid molecule as defined in claim 14.
The composition of claim 39 wherein:

The first nucleic acid is a nucleotide sequence encoding the protein of claim 5 or a fusion protein comprising the protein;

The isotropic repeat is an isolated nucleic acid molecule as defined in claim 15.
The composition of claim 39 wherein:

The first nucleic acid is a nucleotide sequence encoding the protein of claim 6 or a fusion protein comprising the protein;

The isotropic repeat is an isolated nucleic acid molecule as defined in claim 16.
The composition of claim 39 wherein:

The first nucleic acid is a nucleotide sequence encoding the protein of claim 7 or a fusion protein comprising the protein;

The isotropic repeat is an isolated nucleic acid molecule as defined in claim 17.
The composition of claim 39 wherein:

The first nucleic acid is a nucleotide sequence encoding the protein of claim 8 or a fusion protein comprising the protein;

The isotropic repeat is an isolated nucleic acid molecule as defined in claim 18.
The composition of claim 39 wherein:

The first nucleic acid is a nucleotide sequence encoding the protein of claim 9 or a fusion protein comprising the protein;

The isotropic repeat sequence is an isolated nucleic acid molecule as defined in claim 19.
The composition of any one of claims 31 to 46, wherein the target sequence is an RNA sequence derived from a prokaryotic cell or a eukaryotic cell; or the target sequence is a non-naturally occurring RNA sequence.
The composition of any one of claims 31 to 47, wherein the target sequence is present in a cell;

For example, the target sequence is present in the nucleus or in the cytoplasm (eg, organelle);

For example, the cell is a prokaryotic cell.
The composition of any one of claims 31-48, wherein the protein is linked to one or more NLS sequences, or the conjugate or fusion protein comprises one or more NLS sequences;

For example, the NLS sequence is linked to the N-terminus or C-terminus of the protein;

For example, the NLS sequence is fused to the N-terminus or C-terminus of the protein.
A kit comprising one or more components selected from the group consisting of the protein of any one of claims 1-9, the conjugate of claim 10, and the fusion protein of claim 11. The isolated nucleic acid molecule according to any one of claims 12 to 19, the complex according to any one of claims 20 to 27, the isolated nucleic acid molecule according to claim 28, the vector according to claim 29, The composition of any of claims 31-49;

For example, the kit comprises the composition of any of claims 31-38, and instructions for using the composition;

For example, the kit comprises the composition of any of claims 39-46, and instructions for using the composition.
A delivery composition comprising a delivery vehicle, and one or more selected from the group consisting of the protein of any of claims 1-9, the conjugate of claim 10, and the method of claim 11. A fusion protein, the isolated nucleic acid molecule of any one of claims 12 to 19, the complex of any one of claims 20 to 27, the isolated nucleic acid molecule of claim 28, or the method of claim 29. A carrier according to any one of claims 31 to 49;

For example, the delivery vehicle is a particle;

For example, the delivery vehicle is selected from the group consisting of 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 (eg, a replication-defective retrovirus, a lentivirus, Adenovirus or adeno-associated virus).
A method of modifying a target sequence, comprising: combining the complex of any one of claims 20-27, the composition of any one of claims 31-49, or the delivery composition of claim 51 The target sequence is contacted or delivered to a cell comprising the target sequence; wherein the target sequence is associated with or is present in the gene of interest, and the target sequence is RNA;

For example, the target sequence is present in a cell;

For example, the cell is a prokaryotic cell;

For example, the target sequence is present in a nucleic acid molecule (eg, a plasmid) in vitro;

For example, the modification refers to a cleavage of the target sequence, such as a double-strand break or a single-strand break;

For example, the modification further comprises inserting an exogenous nucleic acid into the fragment;

For example, the target sequence is an ssRNA.
The method of claim 52, comprising contacting the complex of claim 21, the composition of claim 32, or the composition of claim 40 with the target sequence, or delivering to the In the cells of the target sequence.
The method of claim 52, comprising contacting the complex of claim 22, the composition of claim 33, or the composition of claim 41 with the target sequence, or delivering to the In the cells of the target sequence.
The method of claim 52, comprising contacting the complex of claim 23, the composition of claim 34, or the composition of claim 42 with the target sequence, or delivering to the In the cells of the target sequence.
The method of claim 52, comprising contacting the complex of claim 24, the composition of claim 35, or the composition of claim 43 with the target sequence, or delivering to the inclusion of the In the cells of the target sequence.
The method of claim 52, comprising contacting the complex of claim 25, the composition of claim 36, or the composition of claim 44 with the target sequence, or delivering to the In the cells of the target sequence.
The method of claim 52, comprising contacting the complex of claim 26, the composition of claim 37, or the composition of claim 45 with the target sequence, or delivering to the In the cells of the target sequence.
The method of claim 52, comprising contacting the complex of claim 27, the composition of claim 38, or the composition of claim 46 with the target sequence, or delivering to the inclusion In the cells of the target sequence.
The method of any one of claims 52-59, wherein the protein, conjugate, fusion protein, isolated nucleic acid molecule, complex, vector or composition is included in a delivery vehicle;

For example, the delivery vehicle is selected from the group consisting of lipid particles, sugar particles, metal particles, protein particles, liposomes, exosomes, viral vectors (eg, replication defective retroviruses, lentiviruses, adenoviruses, or adeno-associated viruses) .
The method of any of claims 52-60 for use in RNA interference or regulation of gene expression.
The protein of any one of claims 1-9, the conjugate of claim 10, the fusion protein of claim 11, the isolated nucleic acid molecule of any of claims 12-19, the claims The complex of any one of claims 20 to 27, the isolated nucleic acid molecule of claim 28, the vector of claim 29, the composition of any one of claims 31-49, or the method of claim 50 A kit, for use in one or more of the following, or in the preparation of a formulation for one or more selected from the group consisting of:

(1) modifying the target sequence;

(2) RNA interference; or

(3) Regulate gene expression.
A cell or a progeny thereof obtained by the method of any one of claims 52-61, wherein the cell comprises a modification that is not found in its wild type;

For example, the modification results in a change in transcription or translation of at least one RNA product;

For example, the modification results in increased expression of at least one RNA product;

For example, the modification results in decreased expression of at least one RNA product;

For example, the cell is a prokaryotic cell.
The product of the cell of claim 63 or a progeny thereof.
An in vitro, ex vivo or in vivo cell or cell line or a progeny thereof, the cell or cell line or a progeny thereof comprising: the protein of any one of claims 1-9, claim 10 The conjugate, the fusion protein of claim 11, the isolated nucleic acid molecule of any one of claims 12 to 19, the complex of any one of claims 20-27, and the method of claim 28. An isolated nucleic acid molecule, the vector of claim 29, the composition of any one of claims 31-49;

For example, the cell or cell line or a progeny thereof comprises: the complex of claim 21, the composition of claim 32 or the composition of claim 40;

For example, the cell or cell line or a progeny thereof comprises: the complex of claim 22, the composition of claim 33 or the composition of claim 41;

For example, the cell or cell line or a progeny thereof comprises: the complex of claim 23, the composition of claim 34 or the composition of claim 42;

For example, the cell or cell line or a progeny thereof comprises: the complex of claim 24, the composition of claim 35 or the composition of claim 43;

For example, the cell or cell line or a progeny thereof comprises: the complex of claim 25, the composition of claim 36 or the composition of claim 44;

For example, the cell or cell line or a progeny thereof comprises: the complex of claim 26, the composition of claim 37 or the composition of claim 45;

For example, the cell or cell line or a progeny thereof comprises: the complex of claim 27, the composition of claim 38 or the composition of claim 46;

For example, the cell is a eukaryotic cell;

For example, the cell is an animal cell (eg, a mammalian cell, such as a human cell) or a plant cell;

For example, the cell is a stem cell or stem cell line.
A method of detecting a target sequence, comprising the composition of any one of claims 20-27, the composition of any one of claims 31-49, or the delivery composition of claim 51 The target sequence is contacted or delivered to a cell comprising the target sequence; wherein the target sequence is RNA;

For example, a targeting sequence contained in the complex, composition or delivery composition is capable of hybridizing to the target sequence;

For example, the target sequence is present in a nucleic acid molecule in vitro;

For example, the target sequence is present in a cell;

For example, the cell is a living cell;

For example, the protein component comprised by the complex, composition or delivery composition carries a detectable label;

For example, the protein component comprised by the complex, composition or delivery composition is fused with a fluorescent protein (eg GFP);

For example, the method is northern blot imprinting;

For example, the method is fluorescence in situ hybridization.
The protein of any one of claims 1-9, the conjugate of claim 10, the fusion protein of claim 11, the isolated nucleic acid molecule of any of claims 12-19, the claims The complex of any one of claims 20 to 27, the isolated nucleic acid molecule of claim 28, the vector of claim 29, the composition of any one of claims 31-49, and the method of claim 50. A kit or a delivery composition according to claim 51 for use in detecting a target sequence or in the preparation of a preparation for detecting a target sequence.
A method of modulating the state of a target cell, comprising the composition of any one of claims 20-27, the composition of any one of claims 31-49, or the delivery composition of claim 51 Introduced into the target cell;

For example, a target sequence that hybridizes to a targeting sequence contained in the complex, composition or delivery composition is present in the target cell;

For example, the state of regulating the target cell comprises:

(1) inducing cell dormancy in vitro or in vivo;

(2) Inducing cell cycle arrest in vitro or in vivo;

(3) inhibiting cell growth and/or cell proliferation in vitro or in vivo;

(4) inducing cell anergy in vitro or in vivo;

(5) inducing apoptosis in vitro or in vivo;

(6) inducing cell necrosis in vitro or in vivo;

(7) inducing cell death in vitro or in vivo; or

(8) Inducing programmed cell death in vitro or in vivo.
The protein of any one of claims 1-9, the conjugate of claim 10, the fusion protein of claim 11, the isolated nucleic acid molecule of any of claims 12-19, the claims The complex of any one of claims 20 to 27, the isolated nucleic acid molecule of claim 28, the vector of claim 29, the composition of any one of claims 31-49, and the method of claim 50. Kit or the delivery composition of claim 51, for use in one or more of the following, or in the preparation of a formulation for one or more selected from the group consisting of :

(1) inducing cell dormancy in vitro or in vivo;

(2) Inducing cell cycle arrest in vitro or in vivo;

(3) inhibiting cell growth and/or cell proliferation in vitro or in vivo;

(4) inducing cell anergy in vitro or in vivo;

(5) inducing apoptosis in vitro or in vivo;

(6) inducing cell necrosis in vitro or in vivo;

(7) inducing cell death in vitro or in vivo; or

(8) Inducing programmed cell death in vitro or in vivo.