CN117327679A - Base editing tool and application thereof - Google Patents

Abstract

The invention belongs to the field of nucleic acid editing, in particular to the technical field of regularly clustered interval short palindromic repeat (CRISPR). Specifically, the invention provides a fusion protein, which comprises a Cas protein and deaminase, wherein the Cas protein in the fusion protein is a Cas mutant protein with inactivated nuclease activity. The invention fuses the Cas protein with deaminase through improving the Cas protein, can be used for single base editing of target nucleic acid, and has wide application prospect.

Description

Base editing tool and application thereof

The present application claims priority from chinese patent application CN202211451897.0, whose application date is 2022, 11, 21. The present application refers to the entirety of the above-mentioned chinese patent.

Technical Field

The invention relates to the field of gene editing, in particular to the technical field of regular clustered interval short palindromic repeat (CRISPR). In particular, the invention relates to a base editing tool, in particular a Cas12 protein-based base editing tool.

Background

CRISPR/Cas technology is a widely used gene editing technology that uses RNA-guided specific binding of target sequences on the genome and cleavage of DNA to create double strand breaks, site-directed gene editing using biological non-homologous end joining or homologous recombination.

The development of single base gene editing tools can realize the accurate modification of specific gene loci without causing DNA double strand breaks. The basic principle of single base gene editing technology is that cytosine deaminase (apodec) or adenosine deaminase is fused with Cas protein to form, and the single base of a target point is modified by relying on CRISPR principle.

The base editor in a single base gene editing system is mainly composed of two parts: cas proteins and DNA modifying enzymes. To date, two types of base editors have been developed: cytosine Base Editors (CBEs) based on cytosine deaminase to effect conversion of C > T (from C to T); the conversion of A > G (from A to G) is achieved based on the Adenine Base Editor (ABE) of adenosine deaminase.

Currently, the Cas enzyme commonly used in single base gene editing tools is Cas9nickase (Cas 9 n) with nickase activity; the single base editing tool capable of base editing is constructed based on the Cas12i protein, single base editing by using the Cas12i protein is realized for the first time, and the application range of the single base editing technology is greatly expanded.

Disclosure of Invention

In one aspect, the invention provides a Cas mutein with inactivated nuclease activity.

In one embodiment, the Cas mutein is mutated at amino acid position 7 and at amino acid position 619 or at amino acid position 844, respectively, corresponding to the amino acid sequence shown in SEQ ID No.1, as compared to the amino acid sequence of the parent Cas protein.

In one embodiment, the Cas mutein is mutated at amino acid positions 7 and 619 corresponding to the amino acid sequence set forth in SEQ ID No.1, as compared to the amino acid sequence of the parent Cas protein.

In one embodiment, the Cas mutein is mutated at amino acid positions 7 and 844 corresponding to the amino acid sequence shown in SEQ ID No.1, as compared to the amino acid sequence of the parent Cas protein.

In one embodiment, the Cas mutein is mutated at amino acid positions 7, 619 and 844 in comparison to the amino acid sequence of the parent Cas protein, corresponding to the amino acid sequence shown in SEQ ID No. 1.

In one embodiment, the Cas mutein further comprises mutations at amino acid positions 233, 267, 369 and 433 corresponding to the amino acid sequence shown in SEQ ID No.1, as compared to the amino acid sequence of the parent Cas protein.

In one embodiment, the Cas mutein is mutated at amino acid position 7 and at amino acid position 619 or at amino acid position 844, respectively, corresponding to the amino acid sequence shown in SEQ ID No.1, compared to the amino acid sequence of the parent Cas protein; further, the Cas mutant protein has mutations at amino acid positions 233, 267, 369 and 433 corresponding to the amino acid sequence shown in SEQ ID No.1, as compared with the amino acid sequence of the parent Cas protein.

In one embodiment, the Cas mutein has mutations at amino acid positions 7, 619, 233, 267, 369 and 433 corresponding to the amino acid sequence shown in SEQ ID No.1, as compared to the amino acid sequence of the parent Cas protein.

In one embodiment, the amino acid at position 7 is mutated to a non-S amino acid, e.g., a, V, G, L, Q, F, W, Y, D, N, E, K, M, T, C, P, H, R, I; preferably, R.

In one embodiment, the amino acid at position 233 or 267 is mutated to an amino acid other than D, e.g., a, V, G, L, Q, F, W, Y, N, S, E, K, M, T, C, P, H, R, I; preferably, the amino acid at position 233 or the amino acid at position 267 is mutated to R.

In one embodiment, the amino acid at position 369 is mutated to a non-N amino acid, e.g., a, V, G, L, Q, F, W, Y, D, S, E, K, M, T, C, P, H, R, I; preferably, R.

In one embodiment, the amino acid at position 433 is mutated to a non-S amino acid, e.g., a, V, G, L, Q, F, W, Y, D, N, E, K, M, T, C, P, H, R, I; preferably, R.

In one embodiment, the 619 amino acid mutation to a non-D amino acid, for example, A, V, G, L, Q, F, W, Y, N, S, E, K, M, T, C, P, H, R, I; preferably, A.

In one embodiment, the 844 amino acid is mutated to a non-E amino acid, e.g., a, V, G, L, D, F, W, Y, N, S, Q, T, M, K, C, P, H, R, I; preferably, A.

In one embodiment, the amino acid sequence of the parent Cas protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity compared to SEQ ID No. 1.

In some embodiments, the parent Cas protein is a native wild-type Cas protein; in other embodiments, the parent Cas protein is an engineered Cas protein.

Cas proteins or Cas12i proteins from a variety of organisms may be used as parent Cas proteins, which in some embodiments have nuclease activity. In some embodiments, the parent Cas protein is a nuclease, i.e., cleaves both strands of a target duplex nucleic acid (e.g., duplex DNA). In some embodiments, the parent Cas protein is a nickase, i.e., a single strand that cleaves a target duplex nucleic acid (e.g., duplex DNA).

In one embodiment, the amino acid sequence of the parent Cas protein is shown as SEQ ID No. 1.

In another aspect, the invention provides a fusion protein comprising a Cas protein and a deaminase.

In one embodiment, the Cas protein in the fusion protein is a Cas mutant protein with inactivated nuclease activity as described above.

In one embodiment, the deaminase is selected from any of adenosine deaminase or cytidine deaminase.

In the present invention, an adenosine deaminase, also known as adenine deaminase, catalyzes the hydrolytic deamination of adenine or adenosine. The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) can be from any organism, such as a bacterium. In some embodiments, the adenosine deaminase is a naturally occurring adenosine deaminase, as well as a variant that is mutated but still has adenosine deaminase activity.

In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is derived from Escherichia coli (Escherichia coli), staphylococcus aureus (Staphylococcus aureus), salmonella typhimurium (Salmonella typhi), shiva putrefying (Shewanella putrefaciens), haemophilus influenzae (Haemophilus influenzae), candida crescens (Caulobacter crescentus), or bacillus subtilis (Bacillus subtilis).

In some embodiments, the adenosine deaminase is TadA deaminase. In some embodiments, the TadA deaminase is an escherichia coli TadA deaminase, but also a variant of a TadA deaminase, e.g., tadA7-10, e.g., tadA9.

In one embodiment, the amino acid sequence of the adenosine deaminase has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5%, or at least 99.9% sequence identity compared to SEQ ID No. 2.

SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAH AEIMALRQGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGS LMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRRVFNAQKKAQSSIN(SEQ IDNo.2)。

In a preferred embodiment, the amino acid sequence of the adenosine deaminase is shown in SEQ ID No. 2.

In the present invention, cytidine deaminase, also known as cytosine deaminase, catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine. In some embodiments, the cytidine deaminase catalyzes the hydrolytic deamination of cytosine to uracil. In some embodiments, the cytidine deaminase is a naturally occurring deaminase from an organism, such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism, but retains cytidine deaminase activity.

In another preferred embodiment, the cytosine deaminase comprises apodec. In one embodiment, the apodec is selected from the group consisting of: apodec 1 (A1), apodec 2 (A2), apodec 3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3H, APOBEC4 (A4), activation-induced deaminase (activation induced cytidine deaminase, AID), or a combination thereof.

In another preferred embodiment, the cytidine deaminase comprises CBE2.0, CBE2.1, CBE2.2, CBE2.3, CBE2.4.

In one embodiment, the amino acid sequence of the cytidine deaminase has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5%, or at least 99.9% sequence identity as compared to SEQ ID No. 3.

STDAEYVRIHEKLDIYTFKKQFSNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWVCKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKRRSELSIMFQVKILHTTKSPAV(SEQ ID No.3)。

In a preferred embodiment, the amino acid sequence of the cytidine deaminase is shown as SEQ ID No. 3.

In one embodiment, the Cas protein in the fusion protein is fused to the N-terminus of the deaminase; in other embodiments, the Cas protein in the fusion protein is fused to the C-terminus of the deaminase. In some embodiments, the Cas protein and deaminase are linked by a linker.

In the present invention, linkers may be used to attach any peptide or protein domain of the present invention. In certain embodiments, the linker is a polypeptide. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is an amide-linked carbon-nitrogen bond. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of an aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, acetic acid, alanine, beta-alanine, 3-aminopropionic acid, 4-aminobutyric acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminocaproic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises an amino acid. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a benzene ring. The linker may comprise a functionalized moiety to facilitate attachment of nucleophiles (e.g., thiols, amino groups) from the peptide to the linker. Any electrophile may be used as part of the linker.

In one embodiment, the linker is an XTEN linker, preferably having the amino acid sequence shown in SEQ ID No. 4. SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID No. 4).

In one embodiment, the fusion protein of the invention further comprises a Nuclear Localization Sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In other embodiments, both the N-and C-segments of the fusion protein are linked to NLS.

In some embodiments, the NLS is fused to the N-terminus of the Cas protein. In some embodiments, the NLS is fused to the C-terminus of the Cas protein. In some embodiments, the NLS is fused to the N-terminus of the deaminase. In some embodiments, the NLS is fused to the C-terminus of the deaminase. In some embodiments, the NLS is fused to the fusion protein via one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker.

The Nuclear Localization Sequence (NLS) is known in the art and will be apparent to the skilled person, in some embodiments the sequence of the NLS comprises the amino acid sequence PKKKRKV (SEQ ID No. 5), or KRPAATKKAGQAKKKK (SEQ ID No. 12).

In one embodiment, the deaminase in the fusion protein is a cytidine deaminase; the fusion protein includes a Cas protein and a cytidine deaminase, further, the fusion protein further includes a Uracil Glycosylase Inhibitor (UGI).

The term "uracil glycosylase inhibitor" or "UGI" refers to a protein capable of inhibiting uracil-DNA glycosylase base cleavage repair enzymes.

In some embodiments, the UGI is fused to the N-terminus or the C-terminus of the Cas protein. In one embodiment, the UGI and Cas proteins are fused via a linker; in other embodiments, the UGI and Cas proteins are not fused via a linker. The linker is preferably an XTEN linker.

In some embodiments, the UGI is fused to the N-terminus or the C-terminus of the deaminase. In one embodiment, the UGI and deaminase are fused via a linker; in other embodiments, the UGI and deaminase are not fused via a linker. The linker is preferably an XTEN linker.

In one embodiment, the UGIs may be 1 or more UGIs, for example, may be formed by 2, 3, 4 or more UGIs connected.

In one embodiment, the amino acid sequence of the UGI has at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5%, or at least 99.9% sequence identity compared to SEQ ID No. 6.

TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTS DAPEYKPWALVIQDSNGENKIKML(SEQ ID No.6)。

In a preferred embodiment, the amino acid sequence of the UGI is shown in SEQ ID No. 6.

It will be apparent to those skilled in the art that the structure of a protein may be altered without adversely affecting its activity and functionality, for example, one or more conservative amino acid substitutions may be introduced into the amino acid sequence of the protein without adversely affecting the activity and/or three-dimensional structure of the protein molecule. Examples and embodiments of conservative amino acid substitutions are apparent to those skilled in the art. In particular, the amino acid residue may be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., with a nonpolar amino acid residue, with a polar uncharged amino acid residue, with a basic amino acid residue, with an acidic amino acid residue. Such substituted amino acid residues may or may not be encoded by the genetic code. Conservative substitutions of one amino acid by another belonging to the same group are within the scope of the invention as long as the substitution does not result in inactivation of the biological activity of the protein. Thus, the proteins of the invention may comprise one or more conservative substitutions in the amino acid sequence, which are preferably made according to table 1. In addition, proteins that also contain one or more other non-conservative substitutions are also contemplated by the present invention, provided that the non-conservative substitutions do not significantly affect the desired function and biological activity of the proteins of the present invention.

Conservative amino acid substitutions may be made at one or more predicted nonessential amino acid residues. "nonessential" amino acid residues are amino acid residues that can be altered (deleted, substituted or substituted) without altering the biological activity, whereas "essential" amino acid residues are required for the biological activity. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Amino acid substitutions may be made in non-conserved regions of the Cas muteins or fusion proteins described above. In general, such substitutions are not made to conserved amino acid residues, or amino acid residues that are within a conserved motif, where such residues are required for protein activity. However, it will be appreciated by those skilled in the art that functional variants may have fewer conservative or non-conservative changes in the conserved regions.

TABLE 1

Initial residues	Representative substitution	Preferred substitution
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

It is well known in the art that one or more amino acid residues may be altered (substituted, deleted, truncated or inserted) from the N-and/or C-terminus of a protein while still retaining its functional activity. Thus, proteins that have one or more amino acid residues altered from the N-and/or C-terminus of a Cas mutant protein while retaining their desired functional activity are also within the scope of the invention. These changes may include changes introduced by modern molecular methods such as PCR, including PCR amplification that alters or extends the protein coding sequence by including an amino acid coding sequence in the oligonucleotides used in the PCR amplification.

It will be appreciated that proteins may be altered in a variety of ways, including amino acid substitutions, deletions, truncations and insertions, and that methods for such manipulation are generally known in the art. For example, amino acid sequence variants of the above proteins can be prepared by mutation of DNA. Single or multiple amino acid substitutions, deletions and/or insertions may also be made by other forms of mutagenesis and/or by directed evolution, for example, using known mutagenesis, recombination and/or shuffling (shuffleling) methods, in combination with associated screening methods.

Those skilled in the art will appreciate that these minor amino acid changes in the Cas proteins of the invention may occur (e.g., naturally occurring mutations) or be generated (e.g., using r-DNA technology) without loss of protein function or activity. If these mutations occur in the catalytic domain, active site or other functional domain of the protein, the nature of the polypeptide may be altered, but the polypeptide may retain its activity. Smaller effects can be expected if mutations are present that are not close to the catalytic domain, active site or other functional domain.

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

In the present invention, amino acid residues may be represented by single letters or by three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamine (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), arginine (Arg, R).

The term "AxxB" means that amino acid a at position xx is changed to amino acid B, e.g. D619A means that D at position 619 is mutated to a. When multiple amino acid positions are mutated simultaneously, the expression can be expressed in a similar form as S7R-D619A, for example, S7R-D619A represents that S at position 7 is mutated to R while D at position 619 is mutated to A.

The specific amino acid positions (numbering) within the proteins of the invention are determined by aligning the amino acid sequence of the protein of interest with the sequence of interest (e.g., SEQ ID No. 1) using standard sequence alignment tools, such as by aligning the two sequences using the Smith-Waterman algorithm or using the CLUSTALW2 algorithm, wherein the sequences are considered aligned when the alignment score is highest. The alignment score can be calculated as described in Wilbur, W.J. and Lipman, D.J. (1983) Rapid similarity searches of nucleic acid and protein data banks, proc.Natl. Acad.Sci.USA, 80:726-730. Default parameters are preferably used in the ClustalW2 (1.82) algorithm: protein gap opening penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein/DNA endplay= -1; protein/DNAGAPDIST =4. The position of a specific amino acid within a protein according to the invention is preferably determined by aligning the amino acid sequence of the protein with SEQ ID No.1 using the AlignX program (part of the vectorNTI group) with default parameters (gap opening penalty: 10 g gap extension penalty 0.05) suitable for multiple alignments. The amino acid sequence of any parent Cas protein can be compared and aligned (aligned) with SEQ ID No.1 using software commonly used in the art, such as Clustal Omega, to obtain the amino acid positions in the parent Cas protein corresponding to the amino acid positions defined based on SEQ ID No.1 as described herein.

The fusion protein of the present invention is not limited to the manner of production, and for example, it can be produced by genetic engineering methods (recombinant techniques) or by chemical synthesis methods.

The invention also provides a base editing tool, e.g., a single base editing tool, comprising the fusion protein described above.

Nucleic acid

In another aspect, the invention provides an isolated polynucleotide comprising:

(a) Polynucleotide sequences encoding Cas muteins or fusion proteins of the invention;

alternatively, a polynucleotide complementary to the polynucleotide of (a).

In one embodiment, the nucleotide sequence is codon optimized for expression in a prokaryotic cell. In one embodiment, the nucleotide sequence is codon optimized for expression in eukaryotic cells.

In one embodiment, the cell is an animal cell, e.g., a mammalian cell.

In one embodiment, the cell is a human cell.

In one embodiment, the cell is a plant cell, such as a cell of a cultivated plant (e.g., cassava, maize, sorghum, wheat, or rice), algae, tree, or vegetable.

In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.

Guide RNA (gRNA)

In another aspect, the invention provides a gRNA comprising a first segment and a second segment; the first segment is also known as a "framework region", "protein binding segment", "protein binding sequence", or "Direct Repeat (Direct Repeat) sequence"; the second segment is also referred to as a "targeting sequence of a targeting nucleic acid" or a "targeting segment of a targeting nucleic acid", or a "targeting sequence of a targeting nucleic acid".

The first segment of the gRNA is capable of interacting with the Cas protein in the fusion protein of the invention, thereby forming a complex of Cas protein and gRNA.

The targeting sequence of the targeting nucleic acid or targeting segment of the targeting nucleic acid of the invention comprises a nucleotide sequence complementary to a sequence in the target nucleic acid. In other words, the targeting sequence of the targeting nucleic acid or targeting segment of the targeting nucleic acid of the invention interacts with the target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing). Thus, the targeting sequence of the targeting nucleic acid or targeting segment of the targeting nucleic acid may be altered, or may be modified to hybridize to any desired sequence within the target nucleic acid. The nucleic acid is selected from DNA or RNA.

The targeting sequence of the targeting nucleic acid or the percentage of complementarity between the targeting segment of the targeting nucleic acid and the target sequence of the target nucleic acid can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).

The "framework region", "protein binding segment", "protein binding sequence", or "cognate repeat" of the gRNA of the invention can interact with a CRISPR protein (or Cas protein). The gRNA of the invention directs its interacting Cas protein to a specific nucleotide sequence within the target nucleic acid through the action of the targeting sequence of the targeting nucleic acid.

Preferably, the guide RNA comprises a first segment and a second segment in the 5 'to 3' direction.

In the context of the present invention, the second segment is also understood as a guide sequence which hybridizes to the target sequence.

The gRNA of the invention is capable of forming a complex with the Cas protein.

Carrier body

The invention also provides a vector comprising a Cas mutein, fusion protein, isolated nucleic acid molecule or polynucleotide as described above; preferably, it further comprises a regulatory element operably linked thereto.

In one embodiment, the regulatory element is selected from one or more of the following group: enhancers, transposons, promoters, terminators, leader sequences, polyadenylation sequences, and marker genes.

In one embodiment, the vector comprises a cloning vector, an expression vector, a shuttle vector, an integration vector.

In some embodiments, the vectors included in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), but may also be of the plasmid, viral, cosmid, phage, etc. type, which are well known to those skilled in the art.

CRISPR system

The present invention provides an engineered non-naturally occurring vector system, or CRISPR-Cas system, comprising the fusion protein described above or a nucleic acid sequence encoding said fusion protein and a nucleic acid encoding one or more of the guide RNAs described above.

In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid encoding one or more guide RNAs are synthetic.

In one embodiment, the nucleic acid sequence encoding the fusion protein and the nucleic acid encoding one or more guide RNAs do not co-occur naturally.

The one or more guide RNAs target one or more target sequences in the cell. The one or more target sequences hybridize to a genomic locus of a DNA molecule encoding one or more gene products and the fusion protein is directed to the genomic locus of the DNA molecule of the one or more gene products, and the target sequence is modified, edited after the fusion protein reaches the target sequence position, whereby expression of the one or more gene products is altered or modified.

The cells of the invention include one or more of animals, plants or microorganisms.

In some embodiments, the fusion protein is codon optimized for expression in a cell.

The invention also provides an engineered non-naturally occurring carrier system that can include one or more carriers comprising:

a) A first regulatory element operably linked to the gRNA,

b) A second regulatory element operably linked to the fusion protein;

wherein components (a) and (b) are on the same or different supports of the system.

The first and second regulatory elements include promoters (e.g., constitutive promoters or inducible promoters), enhancers (e.g., 35S promoter or 35S enhanced promoter), internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcriptional termination signals, such as polyadenylation signals and poly U sequences).

In some embodiments, the vectors in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), but may also be of the plasmid, viral, cosmid, phage, etc. type, which are well known to those skilled in the art.

In some embodiments, the systems provided herein are in a delivery system. In some embodiments, the delivery system is a nanoparticle, liposome, exosome, microvesicle, or gene gun.

In one embodiment, the target sequence is a DNA or RNA sequence from a prokaryotic or eukaryotic cell. In one embodiment, the target sequence is a non-naturally occurring DNA or RNA sequence.

In one embodiment, the target sequence is present in a cell. In one embodiment, the target sequence is present in the nucleus or in the cytoplasm (e.g., organelle). In one embodiment, the cell is a eukaryotic cell. In other embodiments, the cell is a prokaryotic cell.

Protein-nucleic acid complexes/compositions

In another aspect, the invention provides a complex or composition comprising:

(i) A protein component selected from the group consisting of: the fusion protein; and

(ii) A nucleic acid component comprising (a) a guide sequence capable of hybridizing to a target sequence; and (b) a homeotropic repeat capable of binding to a Cas protein in a fusion protein of the invention.

The protein component and the nucleic acid component are capable of binding to each other to form a complex.

In one embodiment, the nucleic acid component is a guide RNA in a CRISPR-Cas system.

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

Delivery and delivery compositions

The fusion proteins, grnas, nucleic acid molecules, vectors, systems, complexes, and compositions of the invention may be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipofection, nuclear transfection, microinjection, sonoporation, gene gun, calcium phosphate mediated transfection, cationic transfection, lipofection, dendritic transfection, heat shock transfection, nuclear transfection, magnetic transfection, lipofection, puncture transfection, optical transfection, reagent enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, virosomes, artificial virosomes, and the like.

Accordingly, in another aspect, the present invention provides a delivery composition comprising a delivery vehicle, and one or more selected from any of the following: fusion proteins, grnas, nucleic acid molecules, vectors, systems, complexes, and compositions of the invention.

In one embodiment, the delivery vehicle is a particle.

In one embodiment, 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 microbubble, a gene gun, or a viral vector (e.g., replication defective retrovirus, lentivirus, adenovirus, or adeno-associated virus).

Host cells

The invention also relates to an in vitro, ex vivo or in vivo cell or cell line or their progeny comprising: fusion proteins, nucleic acid molecules, protein-nucleic acid complexes, vectors, delivery compositions of the invention are described.

In certain embodiments, the cell is a prokaryotic cell.

In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a non-human mammalian cell, e.g., a non-human primate, bovine, ovine, porcine, canine, simian, rabbit, rodent (e.g., rat or mouse) cell. In certain embodiments, the cells are non-mammalian eukaryotic cells, such as cells of poultry birds (e.g., chickens), fish, or crustaceans (e.g., clams, shrimps). In certain embodiments, the cell is a plant cell, e.g., a cell of a monocot or dicot or a cell of a cultivated plant or a food crop such as tapioca, corn, sorghum, soybean, wheat, oat, or rice, e.g., an algae, tree, or production plant, fruit, or vegetable (e.g., a tree such as citrus, nut, eggplant, cotton, tobacco, tomato, grape, coffee, cocoa, etc.).

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

In certain instances, a host cell of the invention comprises a modification of a gene or genome that is not present in its wild type.

Gene editing method and application

The fusion proteins, nucleic acids, the above-described compositions, the above-described CIRSPR/Cas systems, the above-described vector systems, the above-described delivery compositions, or the above-described host cells of the present invention may be used for any one or any of several of the following uses: targeting and/or editing a target nucleic acid; editing the double-stranded nucleic acid specifically; base editing double-stranded nucleic acid; base editing single stranded nucleic acids. In other embodiments, it may also be used to prepare reagents or kits for any one or any of several of the uses described above.

The invention also provides a method of editing a nucleic acid comprising the step of contacting a target region of a nucleic acid (e.g., a double stranded DNA sequence) with a complex comprising a fusion protein as described above and a gRNA; wherein the target region comprises a targeted base pair, and base substitution is performed on the targeted base pair in the target region. In one embodiment, the deaminase in the fusion protein is an adenosine deaminase, and the targeted base pair is replaced by A: T to G: C; in one embodiment, the deaminase in the fusion protein is a cytidine deaminase and the targeted base pair is replaced by C: G to A: T.

T is the base paired by the component base pairs, and A and T are the base paired by the component base pairs; similarly, G: C refers to the bases paired by the constituent base pairs as G and C, and C: G refers to the bases paired by the constituent base pairs as C and G.

The invention also provides the use of a fusion protein, a nucleic acid, the composition, the CIRSPR/Cas system, the vector system, the delivery composition or the host cell in gene editing; or in the preparation of a reagent or kit for gene editing.

In one embodiment, the gene editing is performed intracellularly and/or extracellularly.

The invention also provides a method of editing a target nucleic acid comprising contacting the target nucleic acid with the fusion protein, nucleic acid, composition, CIRSPR/Cas system, vector system, or delivery composition described above. In one embodiment, the method is editing the target nucleic acid either intra-or extracellular.

The gene editing or editing target nucleic acid includes the step of editing a single base of a target gene.

The editing may be performed in prokaryotic and/or eukaryotic cells.

In another aspect, the invention also provides a kit for gene editing comprising the fusion protein, gRNA, nucleic acid, composition, CIRSPR/Cas system, vector system, delivery composition or host cell described above.

In another aspect, the invention provides the use of the fusion protein, nucleic acid, composition, CIRSPR/Cas system, vector system, delivery composition or host cell described above in the preparation of a formulation or kit for:

(i) Gene or genome editing;

(ii) Editing a target sequence in a target locus to modify an organism;

(iii) Editing a single base;

(iv) Treatment of disease.

Preferably, the gene or genome editing is performed in or out of a cell.

Preferably, the treatment of the disease is treatment of a condition caused by a defect in the target sequence in the target locus.

Method for specifically modifying target nucleic acid

In another aspect, the invention also provides a method of specifically modifying a target nucleic acid, the method comprising: contacting a target nucleic acid with the fusion protein, nucleic acid, composition, CIRSPR/Cas system, vector system, or delivery composition.

The specific modification may occur in vivo or in vitro.

The specific modification may occur either intracellularly or extracellularly.

In some cases, the cell is selected from a prokaryotic cell or a eukaryotic cell, e.g., an animal cell, a plant cell, or a microbial cell.

CRISPR system

As used herein, the term "regularly clustered, spaced short palindromic repeats (CRISPR) -CRISPR-associated (Cas) (CRISPR-Cas) system" or "CRISPR system" is used interchangeably and has the meaning commonly understood by those skilled in the art, which generally comprises transcripts or other elements related to the expression of a CRISPR-associated ("Cas") gene, or transcripts or other elements capable of directing the activity of the Cas gene. The Cas protein in the invention is Crispr associated protein.

CRISPR/Cas complexes

As used herein, the term "CRISPR/Cas complex" refers to a complex formed by directing RNA (guide RNA) or mature crRNA to bind to a Cas protein, comprising a direct repeat sequence that hybridizes to a target sequence and binds to a Cas protein, which complex is capable of recognizing and cleaving a polynucleotide that hybridizes to the guide RNA or mature crRNA.

Guide RNA (guide RNA, gRNA)

As used herein, the terms "guide RNA", "mature crRNA", "guide sequence" are used interchangeably and have the meaning commonly understood by those skilled in the art. In general, the guide RNA can comprise, consist essentially of, or consist of a direct repeat (direct repeat) and a guide sequence.

In certain instances, the guide sequence is any polynucleotide sequence that has sufficient complementarity to a target sequence to hybridize to the target sequence and direct specific binding of the CRISPR/Cas complex to the target sequence. In one embodiment, the degree of complementarity between a guide sequence and its corresponding target sequence is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% when optimally aligned. It is within the ability of one of ordinary skill in the art to determine the optimal alignment. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, the Smith-Waterman algorithm (Smith-Waterman), bowtie, geneious, biopython, and SeqMan in ClustalW, matlab.

Target sequence

"target sequence" refers to a polynucleotide targeted by a guide sequence in a gRNA, e.g., a sequence that has complementarity to the guide sequence, wherein hybridization between the target sequence and the guide sequence will promote the formation of a CRISPR/Cas complex (including Cas proteins and grnas). Complete complementarity is not necessary so long as sufficient complementarity exists to cause hybridization and promote the formation of a CRISPR/Cas complex.

The target sequence may comprise any polynucleotide, such as DNA or RNA. In some cases, the target sequence is located either inside or outside the cell. In some cases, the target sequence is located in the nucleus or cytoplasm of the cell. In some cases, the target sequence may be located within an organelle of a eukaryotic cell, such as a mitochondria or chloroplast. Sequences or templates that can be used for recombination into a target locus comprising the target sequence are referred to as "editing templates" or "editing polynucleotides" or "editing sequences". In one embodiment, the editing template is an exogenous nucleic acid. In one embodiment, the recombination is homologous recombination.

In the present invention, a "target sequence" or "target polynucleotide" or "target nucleic acid" may be any polynucleotide that is endogenous or exogenous to a cell (e.g., a eukaryotic cell). For example, the target polynucleotide may be a polynucleotide that is present in the nucleus of a eukaryotic cell. The target polynucleotide may be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or unwanted DNA). In some cases, the target sequence should be related to the Protospacer Adjacent Motif (PAM).

Wild type

As used herein, the term "wild-type" has the meaning commonly understood by those skilled in the art, which refers to a typical form of an organism, strain, gene, or a characteristic that, when it exists in nature, differs from a mutant or variant form, which may be isolated from a source in nature and not intentionally modified by man.

Non-naturally occurring

As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and refer to human involvement. When these terms are used to describe a nucleic acid molecule or polypeptide, it means that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component to which it is associated in nature or as found in nature.

Identity of

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be conveniently performed using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.48: 443-453. The percent identity between two amino acid sequences can also be determined using the algorithms of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988)) which have been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithm that has been incorporated into the GAP program of the GCG software package (available on www.gcg.com), using the Blossum 62 matrix or PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

Carrier body

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule linked thereto. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; a nucleic acid molecule comprising one or more free ends, free ends (e.g., circular); a nucleic acid molecule comprising DNA, RNA, or both; and other diverse polynucleotides known in the art. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. A vector may be introduced into a host cell to thereby produce a transcript, protein, or peptide, including from a protein, fusion protein, isolated nucleic acid molecule, or the like (e.g., a CRISPR transcript, such as a nucleic acid transcript, protein, or enzyme) as described herein. A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA fragments may 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 in a vector used to package a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus). Viral vectors also comprise polynucleotides carried by a virus for transfection into a host cell. Certain vectors (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced.

Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors".

Host cells

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as e.g. escherichia coli or bacillus subtilis, eukaryotic cells such as microbial cells, fungal cells, animal cells and plant cells.

Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the desired level of expression, and the like.

Regulatory element

As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly U sequences), the detailed description of which may be found in goldel (Goeddel), gene expression techniques: methods of enzymology (GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY) 185, academic Press (Academic Press), san Diego (San Diego), calif. (1990). In some cases, regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells as well as those sequences that direct expression of the nucleotide sequence in only certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may primarily direct expression in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). In some cases, regulatory elements may also direct expression in a time-dependent manner (e.g., in a cell cycle-dependent or developmental stage-dependent manner), which may or may not be tissue or cell type specific. In certain instances, the term "regulatory element" encompasses enhancer elements, such as WPRE; a CMV enhancer; the R-U5' fragment in the LTR of HTLV-I (mol. Cell. Biol., volume 8 (1), pages 466-472, 1988), the SV40 enhancer, and the intron sequence between exons 2 and 3 of rabbit beta-globin (Proc. Natl. Acad. Sci. USA., volume 78 (3), pages 1527-31, 1981).

Promoters

As used herein, the term "promoter" has a meaning well known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of a gene that is capable of initiating expression of a downstream gene. Constitutive (constitutive) promoters are nucleotide sequences of: when operably linked to a polynucleotide encoding or defining a gene product, it results in the production of the gene product in the cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence which, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in a cell, essentially only when an inducer corresponding to the promoter is present in the cell. Tissue specific promoters are nucleotide sequences that: when operably linked to a polynucleotide encoding or defining a gene product, it results in the production of the gene product in the cell substantially only if the cell is a cell of the tissue type to which the promoter corresponds.

NLS

A "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" a protein for introduction into the nucleus by nuclear transport, i.e., a protein with NLS is transported to the nucleus. Typically, NLS contains positively charged Lys or Arg residues exposed at the protein surface. Exemplary nuclear localization sequences include, but are not limited to, NLS from: SV40 large T antigen, EGL-13, c-Myc, and TUS proteins. In some embodiments, the NLS comprises PKKKRKV sequence. In some embodiments, the NLS comprises the AVKRPAATKKAGQAKKKKLD sequence. In some embodiments, the NLS comprises the PAAKRVKLD sequence. In some embodiments, the NLS comprises the MSRRRKANPTKLSENAKKLAKEVEN sequence. In some embodiments, the NLS comprises the KLKIKRPVK sequence. Other nuclear localization sequences include, but are not limited to, the acidic M9 domain of hnRNP A1, the sequences KIPIK and PY-NLS in yeast transcription repressor Mat. Alpha.2.

Operatively connected to

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

Complementarity and method of detecting complementary

As used herein, the term "complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by means of a conventional watson-crick or other non-conventional type. Percent complementarity means the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "fully complementary" means that all consecutive residues of one nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in one second nucleic acid sequence. "substantially complementary" as used herein refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions.

Stringent conditions

As used herein, "stringent conditions" for hybridization refers to conditions under which a nucleic acid having complementarity to a target sequence hybridizes predominantly to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are typically sequence-dependent and will vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.

Hybridization

The term "hybridization" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA, DNA) comprises a nucleotide sequence that enables it to bind non-covalently, i.e., form base pairs and/or G/U base pairs with another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to the complementary nucleic acid), "anneal" or "hybridize".

Hybridization requires that the two nucleic acids contain complementary sequences, although there may be mismatches between bases. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. Typically, the hybridizable nucleic acid is 8 nucleotides or more in length (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

It will be appreciated that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to specifically hybridize. Polynucleotides may comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region in a target nucleic acid sequence to which it hybridizes.

Hybridization of the target sequence to the gRNA represents that at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the target sequence and the nucleic acid sequence of the gRNA can hybridize to form a complex; or at least 12, 15, 16, 17, 18, 19, 20, 21, 22 or more bases of the nucleic acid sequence representing the target sequence and the gRNA may be complementarily paired and hybridized to form a complex.

Expression of

As used herein, the term "expression" refers to a process whereby a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and/or a process whereby the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

Joint

As used herein, the term "linker" refers to a linear polypeptide formed from multiple amino acid residues joined by peptide bonds. The linker of the invention may be an amino acid sequence that is synthesized artificially, 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, e.g., holliger, P. Et al (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; poljak, R.J. Et al (1994) Structure 2:1121-1123).

Treatment of

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

A subject

As used herein, the term "subject" includes, but is not limited to, various animals, plants, and microorganisms.

Animals

Such as mammals, e.g., bovine, equine, ovine, porcine, canine, feline, lagomorph (e.g., mice or rats), non-human primate (e.g., macaque or cynomolgus) or human. In certain embodiments, the subject (e.g., human) has a disorder (e.g., a disorder resulting from a disease-related gene defect).

Plants and methods of making the same

The term "plant" is understood to mean any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops, including artichoke, broccoli, sesame seed, leek, asparagus, lettuce (e.g., head lettuce, leaf lettuce), cabbage (bok choy), yellow arrowroot, melons (e.g., melon, watermelon, columbian melon (crenhaw), white melon, cantaloupe), rape crops (e.g., cabbage, broccoli, chinese cabbage, kohlrabi, chinese cabbage), artichoke, carrot, cabbage (napa), okra, onion, celery, parsley, chick pea, parsnip, chicory, pepper, potato, cucurbit (e.g., zucchini, cucumber, zucchini, melon, pumpkin), radish, dried onion, turnip cabbage, purple eggplant (also known as eggplant), salon, chicory, shallot, chicory, garlic, spinach, green onion, melon, green leafy vegetables (greens), beet (sugar beet and fodder beet), sweet potato, lettuce, horseradish, tomato, turnip, spice; fruit and/or vining crops, such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, oranges, blueberries, boysenberries (boysenberries), redberries, currants, rowfruits, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwi fruits, persimmons, pomegranates, pineapple, tropical fruits, pome fruits, melons, mangoes, papaya, and litchis; field crops, such as clover, alfalfa, evening primrose, white mango, corn/maize (forage maize, sweet maize, popcorn), hops, jojoba, peanuts, rice, safflower, small grain cereal crops (barley, oat, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans), oleaginous plants (rape, mustard, olives, sunflower, coconut, castor oil plants, cocoa beans, groundnut), arabidopsis, fibrous plants (cotton, flax, jute), camphorridae (cinnamon, camphordons), or a plant such as coffee, sugarcane, tea, and natural rubber plants; and/or flower bed plants, such as flowering plants, cactus, fleshy plants and/or ornamental plants, and trees, such as forests (broadleaf and evergreen trees, e.g., conifers), fruit trees, ornamental trees, and nut-bearing trees, and shrubs and other seedlings.

Advantageous effects of the invention

The invention fuses the Cas protein with deaminase through improving the Cas protein, can be used for single base editing of target nucleic acid, and has wide application prospect.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

The sequence information related to the present application is as follows:

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drawings

Figure 1. Results of mutating Cas protein nuclease activity validation.

FIG. 2 is a schematic diagram of the ABE editing tool.

FIG. 3 is a schematic diagram of the CBE editing tool architecture.

FIG. 4. Single base editing efficiency results for Cas proteins based on nuclease activity inactivation; wherein BE4max is the CBE editing vector in the examples, and ABE9 is the ABE editing vector in the examples.

FIG. 5 single base editing efficiency of optimized nuclease activity inactivated Cas protein; wherein S7R-D233R-D267R-N369R-S433R-D619A-aBE is an ABE editing vector formed by the mutant protein S7R-D233R-D267R-N369R-S433R-D619A and adenosine deaminase.

Detailed Description

The following examples are only intended to illustrate the invention and are not intended to limit it. The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. For example, for the conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention, reference may be made to Sambrook (Sambrook), friech (Fritsch) and manitis (Maniatis), molecular cloning: laboratory Manual (MOLECULAR CLONING: A LABORATORY MANUAL), edit 2 (1989); the handbook of contemporary molecular biology (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (edited by f.m. ausubel (f.m. ausubel) et al, (1987)); series (academic publishing company) of methods in enzymology (METHODS IN ENZYMOLOGY): PCR 2: practical methods (PCR 2:A PRACTICAL APPROACH) (m.j. Maxfresen (m.j. Macpherson), b.d. black ms (b.d. hames) and g.r. taylor (1995)), harlow and Lane (Lane) edits (1988), antibodies: laboratory Manual (ANTIBODIES, ALABORATORY Manual), animal cell CULTURE (ANIMAL CELL CULTURE) (R.I. French Lei Xieni (R.I. Freshney) eds. (1987)).

In addition, the specific conditions are not specified in the examples, and the process is carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1 acquisition of nuclease Activity-inactivated Cas proteins

For the known Cas protein (cas12f.4 in CN111757889B, in this example, it is called Cas12i 3), the amino acid sequence of the wild-type Cas12i3 protein is shown in SEQ ID No. 1. Applicants have obtained mutant proteins D619A and E844A of Cas12i3 by bioinformatically predicting the key amino acid positions that may affect their biological functions, and mutating the amino acid positions, D619A to a (D619A), E844A to a (E844A). Mutations at amino acid positions can be used to generate variants of Cas proteins by PCR-based site-directed mutagenesis, which can be performed in a site-directed mutagenesis manner common in the art. The specific method is to design a DNA sequence of Cas12i3 protein (sequence shown as SEQ ID No. 1) into two parts by taking a mutation site as a center, design two pairs of primers to amplify the two parts of DNA sequences respectively, and introduce a sequence requiring mutation on the primers. The combination of mutants was constructed by splitting the DNA into multiple fragments and using PCR, gibson clone. Fragment amplification kit: transStart FastPfu DNA Polymerase (containing 2.5mM dNTPs) and the specific experimental procedures are shown in the specification. Glue recovery kit: Gel DNA Extraction Mini Kit, the detailed experimental procedures are shown in the specification. Kit for vector construction: pEASY-Basic Seamless Cloning and Assembly Kit (CU 201-03), the specific experimental procedures are described in the specification.

To verify the nuclease cleavage activity of the above mutated Cas12i3 proteins D619A and E844A, the PCR double-stranded DNA product shown in SEQ id No.11 was used, and the gRNA targeting sequence was designed for the PCR double-stranded product (agagaaugugugcauagucacacaugcagaguucacuuuug).

The experimental method comprises the following steps: cas protein was 50nM final, gRNA was 50nM final, dsDNA was 300ng final, and the samples were incubated with T7 cleavage buffer for 30min and 10ul running gel were spotted for testing.

The results are shown in FIG. 1. Lane 1 is a wild-type Cas12i3 experimental group that produces significant cleavage activity on double-stranded DNA; lane 2 is Cas12i3 control without added gRNA, double stranded DNA was not cleaved; lane 3 is a blank (water) control; lane 4 is the D619A panel (gRNA added) with no double stranded DNA cleavage; lane 5 is the E844A experimental group (gRNA added) with no cleavage of double stranded DNA. As can be seen from fig. 1, the D619A and E844A mutant proteins completely lost cleavage activity relative to wild type Cas12i3 (referred to as dCas12i 3), and can be used as Cas proteins for single base editing.

Example 2 establishment of Single base editing System based on dCAS12i3

A single base editing system was constructed using dmas 12i3 (D619A or E844A) with inactivated nuclease activity obtained in example 1 and deaminase (adenosine deaminase or cytidine deaminase).

In this embodiment, tadA9 (the amino acid sequence is shown as SEQ ID No. 2) is adopted as the adenosine deaminase, and BE4max (the amino acid sequence is shown as SEQ ID No. 3) is adopted as the cytidine deaminase; the above-described adenosine deaminase and cytidine deaminase are merely exemplary deaminase, and in other embodiments, other adenosine deaminase and cytidine deaminase may be employed.

A schematic representation of an ABE editing element constructed using the adenosine deaminase TadA9 is shown in FIG. 2, and a schematic representation of a CBE editing element constructed using the cytidine deaminase BE4max is shown in FIG. 3; among them, GFP is a label designed to screen positive cells.

As shown in fig. 2-3, deaminase (adenosine deaminase or cytidine deaminase) is N-terminal to dCas12i3 (D619A or E844A) linked by XTEN linker; the other ends of deaminase and dCas12i3 are also ligated with NLS. In the CBE editing element of cytidine deaminase, UGI is also connected to the C-terminal of dCAS12i3 (dCAS 12i3 and UGI are connected by a linker), in this embodiment 2 UGI are used in tandem. The above are merely exemplary ways to attach deaminase and dCas12i3, and in other embodiments, the positions or order of attachment of the above elements may be adjusted by those skilled in the art.

The amino acid sequence and the DNA sequence of the ABE editing element designed in the embodiment are respectively shown as SEQ ID No.7 and SEQ ID No. 8; the amino acid sequence and the DNA sequence of the designed CBE editing element are shown as SEQ ID No.9 and SEQ ID No.10 respectively. The Cas12i3 in the above sequence is a wild-type sequence, and may be replaced with the D619A or E844A protein, respectively, when in actual use.

The activity of the single base editing system is verified in animal cells, and two target FUT8-6 are designed aiming at Chinese Hamster Ovary (CHO) FUT8 genes: TTGAAGCCAAGCTTCTTGGTGGTTTC，FUT8-3：TTCCA GCCAAGGTTGTGGACGGATCAThe method comprises the steps of carrying out a first treatment on the surface of the Wherein, the italic part is PAM sequence, the underlined area is the targeting area of gRNA; the DR region of the gRNA (the region that binds to Cas protein) is: GUCUAACUGCCAGAGAAUC GUGCCUGCAAUUGGCAC.

In this embodiment, FUT8-6 is used for the target of the CBE vector for both D619A and E844A proteins; the target point of the ABE vector aiming at the D619A protein is FUT8-6; the target of the ABE vector against the E844A protein is FU T8-3.

Inserting an ABE or CBE editing element through enzyme cutting sites XbaI and PstI by using a vector pcDNA3.3; the U6 promoter and the gRNA sequence are inserted through the enzyme cutting site Mfe 1. Promoter EF-1. Alpha. Initiates puromycin resistance gene expression. And (3) paving: CHO cells were plated at a confluence of 70-80% and the number of cells seeded in 12 well plates was 8 x 10 x 4 cells/well. Transfection: plating for 24h for transfection, 6.25. Mu.l Hieff Trans were added to 100. Mu.l opti-MEM ^TM A liposome nucleic acid transfection reagent, and mixing uniformly; mu.l opti-MEM was added with 2.5ug of plasmid and mixed well. Diluted Hieff Trans ^TM The liposome nucleic acid transfection reagent is mixed with the diluted plasmid uniformly and incubated for 20min at room temperature. The incubated mixture is added to the cell-plated medium for transfection. Screening by adding puromycin: puromycin was added 24h after transfection, at a final concentration of 10. Mu.g/ml. Puromycin treatment for 24h was replaced with normal medium and incubation continued for 24h. 48h after transfection, cells with GFP signal were sorted by flow cytometry (FACS) digested with trypsin-EDTA (0.05%).

Extracting DNA, PCR amplifying the vicinity of the editing region, and carrying out hiTOM sequencing: cells were collected after pancreatin digestion and genomic DNA was extracted using the cell/tissue genomic DNA extraction kit (baitaike). Amplifying the region near the target for genomic DNA. And (3) performing hitOMe sequencing on the PCR product, and counting the mutation of A-G in ABE and the mutation of C-T in CBE in the target spots. As shown in FIG. 4, neither the ABE vector nor the CB E vector constructed by using the D619A or E844A proteins, respectively, showed single base editing efficiency; BE4max-i3 (D619A) in FIG. 4 is a CBE vector constructed using the D619A protein, BE4max-i3 (E844A) is a CBE vector constructed using the E844A protein, ABE9-i3 (D619A) is an ABE vector constructed using the D619A protein, and ABE9-i3 (E844A) is an ABE vector constructed using the E844A protein; this is probably due to the fact that the mutation of the two unit sites described above, although resulting in a nuclease-inactivated Cas protein, is not sufficient to render it single base editing active when fused to an adenosine deaminase or cytidine deaminase.

In order to obtain Cas protein with single base editing efficiency, the applicant further optimizes Cas protein, specifically, on the basis of D619A or E844A protein, the 7 th amino acid of SEQ ID No.1 is mutated to R, two amino acid site mutated proteins are respectively obtained, S7R-D619A (relative to SEQ ID No.1, the 7 th amino acid is mutated to R, the 619 th amino acid is mutated to A) and S7R-E844A (relative to SEQ ID No.1, the 7 th amino acid is mutated to R, and the 844 th amino acid is mutated to A).

In the same manner as described above, the single base editing efficiency of the S7R-D619A or S7R-E844A protein when used in combination with adenosine deaminase or cytidine deaminase was further verified; as a result, as shown in FIG. 4, the S7R-D619A or S7R-E844A proteins showed significant single base editing activity in both the ABE vector and the CBE vector; BE4max-i3 (S7R-D619A) in FIG. 4 is a CBE vector constructed using the S7R-D619A protein, BE4max-i3 (S7R-E844A) is a CBE vector constructed using the S7R-E844A protein, ABE9-i3 (S7R-D619A) is an ABE vector constructed using the S7R-D619A protein, and ABE9-i3 (S7R-E844A) is an ABE vector constructed using the S7R-E844A protein.

Sequencing results show that the CBE single base editing vector of S7R-D619A or S7R-E844A edits the C at the 9 th position of the 3' end in the FUT8-6 target (AAGCCAAGCTTCTTGGTGGTTTC) into T; the ABE single base editing vector of S7R-D619A edits the 1 st A of the 3' end in the FUT8-6 target (AAGCCAAGCTTCTTGGTGGTTTC) into G; the ABE single base editing vector of S7R-E844A edits the 16 th A of the 3' end in the FUT8-3 target (CAGCCAAGGTTGTGGACGGATCA) into G.

The above results show that base editing tools can be constructed for single base editing using S7R-D619A or S7R-E844A with adenosine deaminase or cytidine deaminase.

Example 3 verification of further optimized Single base editing System

On the basis of the D619A protein obtained in example 1, the amino acid positions at positions 7, 233, 267, 369 and 433 of SEQ ID No.1 were all mutated to R to obtain six amino acid mutated proteins S7R-D233R-D267R-N369R-S433R-D619A (with respect to SEQ ID No.1, amino acid mutation at position 7 to R, amino acid mutation at position 233 to R, amino acid mutation at position 267 to R, amino acid mutation at position 369 to R, amino acid mutation at position 433 to R and amino acid mutation at position 619 to A).

The ABE vector was constructed in the same manner as in example 2, and the single base editing efficiency of the combination of S7R-D233R-D267R-N369R-S433R-D619A with adenosine deaminase was verified, and the gene editing activity was verified in 293T cells, and 3 targets were designed for CHK2, KLF4 and PCSK genes in 293T cells, CHEK2: TGTTTCAACATTGAGAGCTGGGTC; KLF4: GTTTAAACACACCGGGTTAA; PCSK9: CCCAGAGCATCCCGTGGAAC; the average value of the three repeated times of each target point is respectively carried out, the editing efficiency is shown in figure 5, the single base editing efficiency of the S7R-D233R-D267R-N369R-S433R-D619A-ABE vector at the CHK2 target point is 9.15%, the single base editing efficiency at the KLF4 target point is 38.43%, and the single base editing efficiency at the PCSK9 target point is 8.74%.

As can be seen from FIG. 5, the ABE vector composed of S7R-D233R-D267R-N369R-S433R-D619A and adenosine deaminase exhibits excellent single base editing efficiency at different targets, which is higher than that of the S7R-D619A or S7R-E844A proteins of example 2 when used in combination with adenosine deaminase or cytidine deaminase.

In the same manner as in example 2, a CBE vector was constructed using the S7R-D233R-D267R-N369R-S433R-D619A protein, and the editing efficiency of the combination of S7R-D233R-D267R-N369R-S433R-D619A and cytosine deaminase was verified in animal cell CHO cells, and the 3' -terminal 9 th C in FUT8-6 target (AAGCCAAGCTTCTTGGTGGTTTC) was edited into T by using a CBE single base editing vector.

In addition, the applicant also constructed a CBE vector using the S7R-D233R-D267R-N369R-S433R-D619A protein, and single base editing was performed on ALS genes in soybeans, resulting in ALS gene-edited soybeans capable of exhibiting resistance to herbicides.

The above results show that a base editing tool can be constructed for single base editing using S7R-D233R-D267R-N369R-S433R-D619A with an adenosine deaminase or a cytidine deaminase, and that the editing efficiency is higher than that of the S7R-D619A or S7R-E844A protein when used in combination with an adenosine deaminase or a cytidine deaminase.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims (20)

1. A Cas mutein having inactivated nuclease activity, said Cas mutein having a mutation compared to the amino acid sequence of the parent Cas protein at the following amino acid positions corresponding to the amino acid sequence set forth in SEQ ID No. 1:

amino acid position 7, any 1 or 2 amino acid positions selected from amino acid position 619 or amino acid position 844.

2. The Cas mutein of claim 1, wherein the Cas mutein further comprises mutations at amino acid positions 233, 267, 369 and 433 corresponding to the amino acid sequence shown in SEQ ID No.1, as compared to the amino acid sequence of the parent Cas protein.

3. The Cas mutant protein of claim 1, wherein the amino acid sequence of the parent Cas protein is set forth in SEQ ID No. 1.

4. A fusion protein comprising the Cas mutein of any one of claims 1-3 and a deaminase.

5. The fusion protein of claim 4, wherein the deaminase is selected from any one of an adenosine deaminase or a cytidine deaminase.

6. The fusion protein of claim 4 or 5, wherein the Cas mutein and deaminase are linked by a linker.

7. The fusion protein of claim 4 or 5, further comprising a Nuclear Localization Sequence (NLS).

8. The fusion protein of claim 5, wherein the deaminase is a cytidine deaminase and the fusion protein further comprises a Uracil Glycosylase Inhibitor (UGI).

9. An isolated polynucleotide encoding the Cas mutein of any one of claims 1-3 or encoding the fusion protein of any one of claims 4-8.

10. A vector comprising the polynucleotide of claim 9 operably linked to regulatory elements.

11. A CRISPR-Cas system, comprising the fusion protein of any one of claims 4-8 and at least one gRNA;

The gRNA is capable of binding to a Cas mutein in the fusion protein of any one of claims 4-8.

12. A composition, characterized in that it comprises:

(i) A protein component selected from the group consisting of: the Cas mutein of any one of claims 1-3 or the fusion protein of any one of claims 4-8;

(ii) A nucleic acid component that is a gRNA capable of binding to the Cas mutein of any one of claims 1-3.

13. An engineered host cell comprising the Cas mutein of any one of claims 1-3, or the fusion protein of any one of claims 4-8, or the polynucleotide of claim 9, or the vector of claim 10, or the CRISPR-Cas system of claim 11, or the composition of claim 12.

14. Use of the Cas mutein of any one of claims 1-3, or the fusion protein of any one of claims 4-8, or the polynucleotide of claim 9, or the vector of claim 10, or the CRISPR-Cas system of claim 11, or the composition of claim 12, or the host cell of claim 13 in gene editing; or in the preparation of a reagent or kit for gene editing.

15. The use of claim 14, wherein the gene editing is single base editing of the target gene.

16. A kit for gene editing, comprising the Cas mutein of any one of claims 1-3, or the fusion protein of any one of claims 4-8, or the polynucleotide of claim 9, or the vector of claim 10, or the CRISPR-Cas system of claim 11, or the composition of claim 12, or the host cell of claim 13.

17. Use of the Cas mutein of any one of claims 1-3, or the fusion protein of any one of claims 4-8, or the polynucleotide of claim 9, or the vector of claim 10, or the CRISPR-Cas system of claim 11, or the composition of claim 12, or the host cell of claim 13, in the preparation of a formulation or kit for: editing the target sequence in the target locus to modify the organism, or treatment of the disease.

18. A method of editing a nucleic acid, the method comprising the step of contacting a target region of a nucleic acid with the fusion protein of any one of claims 4-8 and a gRNA comprising a segment capable of binding to a Cas mutein in the fusion protein of any one of claims 4-8 and capable of binding to the target region of the nucleic acid; wherein the target region comprises a targeted base pair, the fusion protein being capable of base substitution of the targeted base pair.

19. The method of claim 18, wherein the deaminase in the fusion protein is an adenosine deaminase and the targeted base pair is replaced by a: T to G: C.

20. The method of claim 18, wherein the deaminase in the fusion protein is a cytidine deaminase and the targeted base pair is replaced by C: G to a: T.