阅读说明：本技术 新型CRISPR/Cas12f酶和系统 (Novel CRISPR/Cas12f enzymes and systems ) 是由 赖锦盛 周英思 朱金洁 易飞 张湘博 赵海铭 宋伟彬 于 2019-10-29 设计创作，主要内容包括：本发明属于核酸编辑领域,特别是规律成簇的间隔短回文重复(CRISPR)技术领域。具体而言,本发明提供了Cas效应蛋白、包含此类蛋白的融合蛋白以及编码它们的核酸分子,还提供了包含上述蛋白或核酸分子的用于核酸编辑(例如,基因或基因组编辑)的复合物和组合物,以及包含上述蛋白的用于核酸编辑(例如,基因或基因组编辑)的方法。(The invention belongs to the field of nucleic acid editing, and particularly relates to the technical field of regularly clustered spaced short palindromic repeats (CRISPR). In particular, the invention provides Cas effector proteins, fusion proteins comprising such proteins, and nucleic acid molecules encoding them, as well as complexes and compositions for nucleic acid editing (e.g., gene or genome editing) comprising the above proteins or nucleic acid molecules, and methods for nucleic acid editing (e.g., gene or genome editing) comprising the above proteins.)

A protein having the sequence of SEQ ID NO:1, or an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID No. 1;

for example, the protein is an effector protein in a CRISPR/Cas system.

A conjugate comprising the protein of claim 1 and a modifying moiety.

The conjugate of claim 2, wherein the modifying moiety is selected from the group consisting of an additional protein or polypeptide, a detectable label, and any combination thereof.

The conjugate of claim 2 or 3, wherein the modification moiety is attached to the N-terminus or C-terminus of the protein, optionally via a linker.

The conjugate of any of claims 2-4, wherein the additional protein or polypeptide is selected from an epitope tag, a reporter sequence, a Nuclear Localization Signal (NLS) sequence, a targeting moiety, a transcription activation domain (e.g., VP64), a transcription repression domain (e.g., KRAB domain or SID domain), a nuclease domain (e.g., Fok1), a domain having an activity selected from the group consisting of: nucleotide deaminase, methylase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity and nucleic acid binding activity; and any combination thereof.

The conjugate of any one of claims 2-5, wherein the conjugate comprises an epitope tag.

The conjugate of any one of claims 2-6, wherein the conjugate comprises an NLS sequence;

for example, the NLS sequence is shown as SEQ ID NO: 19;

for example, the NLS sequence is located at, near, or near a terminus of the protein (e.g., N-terminus or C-terminus).

A fusion protein comprising the protein of claim 1 and an additional protein or polypeptide.

The fusion protein of claim 8, wherein the additional protein or polypeptide is linked to the N-terminus or C-terminus of the protein, optionally via a linker.

The fusion protein of claim 8 or 9, wherein the additional protein or polypeptide is selected from an epitope tag, a reporter gene sequence, a Nuclear Localization Signal (NLS) sequence, a targeting moiety, a transcription activation domain (e.g., VP64), a transcription repression domain (e.g., KRAB domain or SID domain), a nuclease domain (e.g., Fok1), a domain having an activity selected from the group consisting of: nucleotide deaminase, methylase activity, demethylase, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity and nucleic acid binding activity; and any combination thereof.

The fusion protein of any one of claims 8-10, wherein the fusion protein comprises an epitope tag.

The fusion protein of any one of claims 8-11, wherein the fusion protein comprises an NLS sequence;

for example, the NLS sequence is shown as SEQ ID NO: 19;

for example, the NLS sequence is located at, near, or near a terminus of the protein (e.g., N-terminus or C-terminus).

The fusion protein of any one of claims 8-12, wherein the fusion protein has the amino acid sequence set forth in SEQ ID No. 20.

An isolated nucleic acid molecule comprising, or consisting of, a sequence selected from the group consisting of:

(i) SEQ ID NO:7 or 13;

(ii) and SEQ ID NO:7 or 13, having substitution, deletion, or addition of one or more bases (e.g., substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases);

(iii) and SEQ ID NO:7 or 13, 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% sequence identity;

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

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

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

for example, the isolated nucleic acid molecule is RNA;

for example, the isolated nucleic acid molecule is a direct repeat in a CRISPR/Cas system.

The isolated nucleic acid molecule of claim 14, wherein the nucleic acid molecule comprises one or more stem loops or optimized secondary structures;

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

The isolated nucleic acid molecule of claim 14 or 15, wherein the nucleic acid molecule comprises or consists of a sequence selected from the group consisting of:

(a) SEQ ID NO:7 or 13;

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

(c) SEQ ID NO:7 or 13, or a complement of the nucleotide sequence set forth in seq id no.

A composite, comprising:

(i) a protein component selected from: the protein of claim 1, the conjugate of any one of claims 2-7, the fusion protein of any one of claims 8-13, and any combination thereof; and

(ii) a nucleic acid component comprising in the 5 'to 3' direction the isolated nucleic acid molecule of any one of claims 14-16 and a targeting sequence capable of hybridizing to a target sequence,

wherein the protein component and the nucleic acid component are bound to each other to form a complex;

for example, the nucleic acid component is a guide RNA in a CRISPR/Cas system;

for example, the nucleic acid molecule is RNA;

for example, the complex does not comprise trans-acting crrna (tracrrna).

The complex of claim 17, wherein said targeting sequence is attached to the 3' end of said nucleic acid molecule.

The complex of claim 17 or 18, wherein the targeting sequence comprises a complementary sequence to the target sequence.

An isolated nucleic acid molecule comprising:

(i) a nucleotide sequence encoding the protein of claim 1, or the fusion protein of any one of claims 8-13;

(ii) a nucleotide sequence encoding the isolated nucleic acid molecule of any one of claims 14-16; and/or the presence of a gas in the gas,

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

for example, the nucleotide sequence described 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 20.

A host cell comprising the isolated nucleic acid molecule of claim 20 or the vector of claim 21.

A composition, comprising:

(i) a first component selected from: the protein of claim 1, the conjugate of any one of claims 2-7, the fusion protein of any one of claims 8-13, a nucleotide sequence encoding the protein or fusion protein, and any combination thereof; and

(ii) a second component which is a nucleotide sequence comprising a guide RNA, or a nucleotide sequence encoding said nucleotide sequence comprising a guide RNA;

wherein the guide RNA comprises a direct repeat sequence and a guide sequence from 5 'to 3' direction, wherein the guide sequence can be hybridized with a target sequence;

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

the direct repeat sequence is an isolated nucleic acid molecule as defined in any one of claims 14 to 16;

for example, the composition does not comprise trans-acting crrna (tracrrna).

A composition comprising one or more carriers comprising:

(i) a first nucleic acid which is a nucleotide sequence encoding the protein of claim 1 or the fusion protein of any one of claims 8-13; optionally the first nucleic acid is operably linked to a first regulatory element; and

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

wherein:

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

the guide RNA comprises a direct repeat sequence and a guide sequence from 5 'to 3' direction, and the guide sequence can be hybridized with a target sequence;

(ii) the guide RNA is capable of forming a complex with the effector protein or fusion protein of (i);

the direct repeat sequence is an isolated nucleic acid molecule as defined in any one of claims 14 to 16;

for example, the composition does not comprise trans-acting crrna (tracrrna).

The composition of claim 24, wherein the first regulatory element and/or the second regulatory element is a promoter, such as an inducible promoter.

The composition of any one of claims 23-25, wherein at least one component of the composition is non-naturally occurring or modified.

The composition of any one of claims 23-26, wherein the targeting sequence is linked to the 3' end of the direct repeat sequence.

The composition of any one of claims 23-27, wherein the targeting sequence comprises a complementary sequence to the target sequence.

The composition of any one of claims 23 to 28, wherein, when the target sequence is DNA, the target sequence is located 3 'of the protospacer adjacent to a motif (PAM), and the PAM has a sequence set forth as 5' -TTN, wherein N is selected from A, G, T, C; when the target sequence is RNA, the target sequence does not have PAM domain restriction.

The composition of any one of claims 23-29, wherein the target sequence is a DNA or RNA sequence from a prokaryotic or eukaryotic cell; alternatively, the target sequence is a non-naturally occurring DNA or RNA sequence.

The composition of any one of claims 23-30, wherein the target sequence is present in a cell;

for example, the target sequence is present in the nucleus or in the cytoplasm (e.g., organelle);

for example, the cell is a eukaryotic cell;

for example, the cell is a prokaryotic cell.

The composition of any one of claims 23-31, wherein the protein has one or more NLS sequences attached thereto, 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 claim 1, the conjugate of any one of claims 2-7, the fusion protein of any one of claims 8-13, the isolated nucleic acid molecule of any one of claims 14-16, the complex of any one of claims 17-19, the isolated nucleic acid molecule of claim 20, the vector of claim 21, the composition of any one of claims 23-32;

for example, the kit comprises the composition of any one of claims 23, 26-32, and instructions for using the composition;

for example, the kit comprises the composition of any one of claims 24, 25-32, 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 claim 1, the conjugate of any one of claims 2-7, the fusion protein of any one of claims 8-13, the isolated nucleic acid molecule of any one of claims 14-16, the complex of any one of claims 17-19, the isolated nucleic acid molecule of claim 20, the vector of claim 21, the composition of any one of claims 23-32;

for example, the delivery vehicle is a particle;

for example, the delivery vector is selected from a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a microvesicle, a gene gun, or a viral vector (e.g., a replication-defective retrovirus, lentivirus, adenovirus, or adeno-associated virus).

A method of modifying a target gene comprising: contacting the complex of any one of claims 17-19 or the composition of any one of claims 23-32 with the target gene, or delivering into a cell comprising the target gene; the target sequence is present in the target gene.

The method of claim 35, wherein the target gene is present in a cell;

for example, the cell is a prokaryotic cell;

for example, the cell is a eukaryotic cell, such as a mammalian cell (e.g., a human cell) or a plant cell.

The method of claim 35, wherein the target gene is present in a nucleic acid molecule (e.g., a plasmid) in vitro.

The method of any one of claims 35-37, wherein the modification is a break in the target sequence, such as a double strand break in DNA or a single strand break in RNA;

for example, the modification further comprises inserting an exogenous nucleic acid into the break.

A method of altering the expression of a gene product comprising: contacting the complex of any one of claims 17-19 or the composition of any one of claims 23-32 with a nucleic acid molecule encoding the gene product, or delivering into a cell comprising the nucleic acid molecule in which the target sequence is present.

The method of claim 39, wherein the nucleic acid molecule is present in a cell;

for example, the cell is a prokaryotic cell;

for example, the cell is a eukaryotic cell, such as a mammalian cell (e.g., a human cell) or a plant cell.

The method of claim 39, wherein the nucleic acid molecule is present in a nucleic acid molecule (e.g., a plasmid) in vitro.

The method of any one of claims 39-41, wherein the expression of the gene product is altered (e.g., enhanced or reduced).

The method of any one of claims 39-42, wherein the gene product is a protein.

The method of any one of claims 35-43, wherein the protein, conjugate, fusion protein, isolated nucleic acid molecule, complex, vector or composition is comprised in a delivery vehicle;

for example, the delivery vector 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 (such as a replication-defective retrovirus, lentivirus, adenovirus, or adeno-associated virus).

The method of any one of claims 35-44, which is used to modify a cell, cell line, or organism by altering one or more target sequences in a target gene or nucleic acid molecule encoding a target gene product.

A cell or progeny thereof obtained by the method of any of claims 35-45, wherein the cell comprises a modification not present in its wild type.

A cell product of the cell of claim 46 or progeny thereof.

An in vitro, ex vivo or in vivo cell or cell line or progeny thereof comprising: the protein of claim 1, the conjugate of any one of claims 2-7, the fusion protein of any one of claims 8-13, the isolated nucleic acid molecule of any one of claims 14-16, the complex of any one of claims 17-19, the isolated nucleic acid molecule of claim 20, the vector of claim 21, the composition of any one of claims 23-32;

for example, the cell is a eukaryotic cell;

for example, the cell is an animal cell (e.g., a mammalian cell, such as a human cell) or a plant cell;

for example, the cell is a stem cell or stem cell line.

The protein of claim 1, the conjugate of any one of claims 2-7, the fusion protein of any one of claims 8-13, the isolated nucleic acid molecule of any one of claims 14-16, the complex of any one of claims 17-19, the isolated nucleic acid molecule of claim 20, the vector of claim 21, the composition of any one of claims 23-32, or the kit of claim 33, for use in nucleic acid editing (e.g., gene or genome editing);

for example, the gene or genome editing comprises modifying a gene, knocking out a gene, altering expression of a gene product, repairing a mutation, and/or inserting a polynucleotide.

Use of the protein of claim 1, the conjugate of any one of claims 2-7, the fusion protein of any one of claims 8-13, the isolated nucleic acid molecule of any one of claims 14-16, the complex of any one of claims 17-19, the isolated nucleic acid molecule of claim 20, the vector of claim 21, the composition of any one of claims 23-32, or the kit of claim 33, in the preparation of a formulation for:

(i) ex vivo gene or genome editing;

(ii) detecting isolated single-stranded DNA;

(iii) editing a target sequence in a target locus to modify an organism or non-human organism;

(iv) treating a condition caused by a defect in a target sequence in a target locus.