Patent Description:
Most human genetic diseases are caused by single base substitution or point mutation rather than some insertions/deletions (indels) or broad chromosomal rearrangement in the genome. Genome editing mediated by programmable nuclease such as clustered, regular interspaced, short palindromic repeat (CRISPR)-Cas9 or Cpf1 system enables gene correction for genetic defects provoking genetic disease, but has a technical difficulty in inducing single base substitution in a target specific manner. This is because most of DNA double-stranded breaks (DSB) generated by programmable nucleases are repaired by error-prone non-homologous end-joining (NHEJ) other than homologous recombination (HR) using a template donor DNA. As a result, indels at nuclease targeted sites occur more frequently than single nucleotide substitutions.

Recently, it has been reported that Cas9 nickase (nCas9) or catalytically-deficient Cas9 (dCas9) linked to cytidine deaminase such as apolipoprotein B editing complex <NUM> (APOBEC1) or activation-induced deaminase (AID) substitutes C for T (or G for A) at target sites without producing DSBs. Such reports exhibit base editing in yeasts and cultured mammalian cells. These RNA-guided programmable deaminases expand the coverage of genome editing to another level and may suggest a method for inducing target mutations or conducting gene editing across all organisms including humans. However, it has to be proved that RNA-programmable deaminases have a base editing function in vivo.

The disclosure provides a technique for inducing a single nucleotide substitution in eukaryotic cells, such as murine cells (e.g., animal cells including mammalian cells but excluding human embryos by using a programmable deaminase.

An embodiment provides a base editing composition comprising: (<NUM>) a deaminase or a coding gene therefor, and (<NUM>) a target-specific nuclease or a coding gene therefor. The cell may be a eukaryotic cell (e.g., a cell from a eukaryotic animal), and the base editing method may be to perform base editing (e.g., base substitution) in a eukaryotic cell.

The target-specific nuclease may include: an RNA-guided nuclease; and a guide RNA hybridizable with a target site in a target gene (having a complementary nucleotide sequence) or a coding DNA therefor (or a recombinant vector carrying the coding DNA). In this regard, the base editing composition may comprise: (<NUM>) a deaminase or a coding gene therefor (mRNA or a recombinant vector carrying the coding DNA), (<NUM>) an RNA-guided nuclease or a coding gene therefor (mRNA or a recombinant vector carrying the coding DNA), and (<NUM>) a guide RNA or a coding gene (DNA) therefor.

The base editing composition may further comprise: (<NUM>) an uracil DNA glycosylase inhibitor (UGI) or a coding gene therefor; and/or (<NUM>) a nuclear localization sequence (NLS) or a coding gene therefor in addition to (<NUM>) a deaminase or a coding gene therefor (mRNA or a recombinant vector carrying the coding DNA), (<NUM>) an RNA-guided nuclease or a coding gene therefor (mRNA or a recombinant vector carrying the coding DNA), and (<NUM>) a guide RNA or a coding gene therefor (DNA). In the case where the base editing composition employs a fusion protein or gene form in which the deaminase, the RNA-guided nuclease, and optionally UGI and/or NLS or coding genes therefor are linked, respectively, a suitable linker may be given between one of the adjacent coupled proteins or genes, for example, between the deaminase and the RNA-guided nuclease, between the nuclease and UGI, and between UGI and NLS (a peptide linker (<NUM>-<NUM> or <NUM>-<NUM> a. ) for the fusion protein and an oligonucleotide linker (<NUM>-<NUM> or <NUM>-<NUM> nt) for the fusion gene).

Another embodiment provides a base editing method comprising a step of introducing the base editing composition into a cell. The cell may be a eukaryotic cell and the base editing method may be to conduct editing (for example, base substitution) in a eukaryotic cell.

According to the invention there is provided a base editing method comprising: directly injecting <NUM>) a ribonucleoprotein complex or mixture, the ribonucleoprotein complex or mixture comprising a cytidine deaminase, an RNA-guided nuclease, and a guide RNA , or <NUM>) a mixture of a cytidine deaminase-encoding mRNA, an RNA-guided <NUM> nuclease-encoding mRNA, and a guide RNA into the non-human mammalian embryonic cell without using a recombinant vector. As stated supra, the base editing composition used in the introducing step conducted through <NUM>) or <NUM>) may further comprise a uracil DNA glycosylase inhibitor (UGI) or a coding gene therefor and/or (<NUM>) a nuclear localization sequence (NLS) or a coding gene therefor, and optionally a suitable linker.

The present inventors successfully induced single nucleotide substitutions in eukaryotic cells such as murine cells (e.g., animal cells such as mammalian cells, etc.) by using a programmable deaminase.

An embodiment provides a base editing composition comprising: (<NUM>) a deaminase or a coding gene therefor, and (<NUM>) a target-specific nuclease or a coding gene therefor. The base editing composition may have base editing (e.g., base substitution) activity in eukaryotic cells. The eukaryotic cells may be cells of eukaryotic animals, for example, non-human embryonic cells. In one embodiment, the eukaryotic cells may be non-human mammalian cells, for example, mammalian embryonic cells.

As used herein, the term "coding gene" is intended to encompass cDNA, rDNA, a recombinant vector carrying the same, and mRNA.

As used herein, the "deaminase" is a generic term for enzymes having the activity of removing an amine group from certain bases in eukaryotic cells and may be, for example, cytidine deaminase, which converts cytidine to uridine, and/or adenosine deaminase. In one embodiment, the deaminase may be at least one selected from the group consisting of apolipoprotein B editing complex <NUM> (APOBEC1), activation-induced deaminase (AID), tRNA-specific adenosine deaminase (tadA), and the like, but is not limited thereto. Single nucleotide substitution in eukaryotic cells can be induced by such base conversion (for example, conversion of cytidine to uridine).

The target-specific nuclease may include an RNA-guided nuclease and a guide RNA capable of hybridizing with (or having a complementary sequence to) a target site of a target gene or a coding DNA therefor (or a recombinant vector carrying the coding DNA). In this context, the base editing composition may comprise: (<NUM>) a deaminase or a coding gene therefor (mRNA or a recombinant vector carrying the coding DNA), (<NUM>) a modified RNA-guided nuclease or a coding gene therefor (mRNA or a recombinant vector carrying the coding DNA), and (<NUM>) a guide RNA or a coding DNA therefor.

In an embodiment, the base editing composition may further comprise: (<NUM>) a uracil DNA glycosylase inhibitor (UGI) or a coding gene therefor; and/or (<NUM>) nuclear localization sequence (NLS) or a coding gene therefor in addition to (<NUM>) a deaminase or a coding gene therefor (mRNA or a recombinant vector carrying the coding DNA), (<NUM>) an RNA-guided nuclease or a coding gene therefor (mRNA or a recombinant vector carrying the coding DNA), and (<NUM>) a guide RNA or a coding gene therefor (DNA). When the base editing composition employs a fusion protein or gene form in which the deaminase, the RNA-guided nuclease, and optionally UGI and/or NLS or coding genes therefor are linked, respectively, a suitable linker may be given between one of the adjacent coupled proteins or genes, for example, between the deaminase and the RNA-guided nuclease, between the nuclease and UGI, and between UGI and NLS (a peptide linker (<NUM>-<NUM> or <NUM>-<NUM> a. ) for the fusion protein and an oligonucleotide linker (<NUM>-<NUM> or <NUM>-<NUM> nt) for the fusion gene).

In an embodiment, the RNA-guided nuclease may be a modified RNA-guided nuclease that is modified to lose the activity of forming DNA double-stranded breaks.

The modified RNA-guided nuclease may be a modified Cas9 (CRISPR associated protein <NUM>) or modified Cpf1 (CRISPR from Prevotella and Francisella <NUM>) system that is modified to cut one strand in a target gene (nick formation). In one embodiment, the modified RNA-guided nuclease may be selected from the group consisting of Cas9 nickase (nCas9) and catalytically-deficient Cas9 (dCas9).

When the base editing composition comprises a deaminase-encoding gene and an RNA-guided nuclease-encoding gene, the encoding gene may be DNA or mRNA. In addition, the deaminase-encoding gene and the RNA-guided nuclease-encoding gene may be included in a form of mRNA or recombinant vectors that carry the DNAs respectively (i.e., one recombinant vector carrying the <NUM> deaminase-encoding DNA and one recombinant vector carrying the RNA guide nuclease-encoding DNA) or a recombinant vector that carries the DNAs together. The guide RNA may be CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), double guide RNA including crRNA and tracrRNA (a complex of crRNA and tracrRNA), or single guide RNA (sgRNA). In an embodiment, the base editing composition may comprise mRNAs coding for a deaminase and a modified RNA-guided nuclease, and a guide RNA, or a ribonucleoprotein (RNP) inclusive of a deaminase, a modified RNA-guided nuclease, and a guide RNA. The ribonucleoprotein may include a deaminase, a modified RNA-guided nuclease, and a guide RNA in mixture or may exist as a complex in which a deaminase, a modified RNA-guided nuclease, and a guide RNA are associated.

Another embodiment provides a base editing method comprising a step of introducing the base editing composition into a cell. The cell may be a eukaryotic cell and the base editing method may be to conduct base editing (e.g., base substitution) in a eukaryotic cell.

The eukaryotic cell may be a eukaryotic animal cell, for example, a eukaryotic non-human embryonic cell. In an embodiment, the cell may be a mammalian cell, for example, a non-human mammalian embryonic cell. The base editing method may achieve a base conversion rate (base substitution rate) of <NUM>% or higher, <NUM>% or higher, <NUM>% or higher, <NUM>% or higher, <NUM>% or higher, <NUM>% or higher, <NUM>% or higher, <NUM>% or higher, <NUM>% or higher, <NUM>% or higher, <NUM>% or higher, <NUM>% or higher, <NUM>% or higher, <NUM>% or higher, or <NUM>% in eukaryotic cells (e.g., eukaryotic non-human embryonic cells). In addition, the base editing method may cause various mutants by base substitution to create a stop codon within a gene (e.g., coding sequence) for gene knockout, to introduce mutations in non-coding DNA sequences that do not encode protein sequences, etc..

Particularly, the base editing composition may be applied to nonhuman mammalian embryos to effectively construct adult mammals having a gene desirably knocked out therein or a desirable mutation introduced thereto.

The step of introducing the base editing composition into a cell may be a step of introducing a deaminase or a deaminase-encoding gene, an RNA-guided nuclease or an RNA-guided nuclease-encoding gene, and a guide RNA or a guide RNA-encoding gene to a cell. Of the coding genes, at least one may be included in respective or one recombinant vector for use in introduction.

In one embodiment, the step of introducing the base editing composition into a cell may be conducted by.

The eukaryotic animals may be mammals including primates, such as humans, (excluding human embryos) and rodents, such as mice, etc. The cells from eukaryotic animals may be non-human mammalian embryos. For example, the embryos may be taken from the oviduct of a superovulated female mammal (superovulated by injection of gonadal hormones, such as PMSG (Pregnant Mare Serum Gonadotropin), hCG (human Choirinic Gonadotropin), and the like) after crossing the superovulated female animals with male mammals. The non-human embryo to which the base editing composition is applied (injected) may be a one-cell zygote at fertilization.

As used herein, the term "base editing" means base mutation (substitution, deletion, or insertion) incurring point mutation at a target site within a target gene and can be distinguished from gene editing in terms of the scale of mutated bases, a small number of mutated bases (one or two bases, i.e., one base) in base editing and relatively many mutated bases in gene editing. The base editing may not result in double-stranded DNA cleavage.

As used herein, the term "base mutation (or base substitution)" means mutation (e.g., substitution) on a nucleotide inclusive of the corresponding base and may be used interchangeably with "nucleotide mutation (or nucleotide substitution)". The base mutation may occur on either or both of allele genes.

In one embodiment, the base mutation and the base editing resulting therefrom may be conducted in various manners including creating a stop codon or a codon accounting for a different amino acid from a wild-type amino acid on a target site to knock a target gene out, or introducing a mutation in a non-coding DNA sequence, which does not encode a protein sequence, etc., but is not limited thereto.

In the disclosure, the base editing or base mutation may be conducted in vitro or in vivo, where the in vivo method is not a method of treatment The term "base sequence", as used herein, means a nucleotide sequence including corresponding bases and can be used interchangeably with a nucleotide sequence or a nucleic acid sequence.

In one embodiment, when the target-specific nuclease includes an RNA-guided nuclease, a guide RNA containing a targeting sequence may be included together with the RNA-guided nuclease. The "targeting sequence" may be a guide RNA site including a base sequence complementary to (hybridizable with) a consecutive base sequence of about <NUM> to about <NUM> nucleotides (nt), about <NUM> to about <NUM> nt, about <NUM> to about <NUM> nt, or about <NUM> to about <NUM> nt, e.g., about <NUM> nt on a target site. The base sequence on a target site, complementary to the targeting sequence, is called a "target sequence". The "target sequence" may mean a consecutive base sequence of about <NUM> nt to about <NUM> nt, about <NUM> nt to about <NUM> nt, about <NUM> nt to about <NUM> nt, or about <NUM> nt to about <NUM> nt, for example, about <NUM> nt located adjacent to the <NUM>'- and/or <NUM>'-end of a PAM sequence recognized by an RNA-guided nuclease.

A deaminase is a generic name for enzymes having the activity of removing an amine group from certain bases in eukaryotic cells, as exemplified by cytidine deaminase and/or adenosine deaminase that converts cytidine to uridine. In one embodiment, the deaminase may be at least one selected from the group consisting of APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like), AID (activation-induced deaminase), and tadA (tRNA-specific adenosine deaminase), but is not limited thereto. APOBEC1, AID, and tadA may be derived from prokaryotic animals such as E. coli, or eukaryotic animals, such as mammals, for example, primates including humans, rodents including mice, etc..

In one embodiment, the APOBEC may be at least one selected from the group consisting of APOBEC1 (apolipoprotein B editing complex <NUM>), APOBEC2, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3E, APOBEC3F, APOBEC3G, APOBEC3H, and APOBEC4, which are all derived from mammals, for example, humans, rats, mice, etc..

The APOBEC1 may be at least one selected from the group consisting of human APOBEC1 (e.g., GenBank Accession No. NP_005880. <NUM> (coding gene: NM_005889. <NUM>), NP_001291495. <NUM> (coding gene: NM_001304566. <NUM>), NP_001635. <NUM> (coding gene: NM_001644. <NUM>), etc.), murine APOBEC1 (e.g., GenBank Accession No. NP_001127863. <NUM> (coding gene: NM_001134391. <NUM>), NP_112436. <NUM> (coding gene: NM_031159. <NUM>), etc.), and rat APOBEC1 (e.g., GenBank Accession No. NP_037039. <NUM> (SEQ ID NO: <NUM>) (coding gene: NM_012907. <NUM>), etc.) and is not limited thereto.

The AID may be selected from the group consisting of human AID (e.g., GenBank Accession No. NP_001317272. <NUM> (coding gene: NM_001330343. <NUM>), NP_065712. <NUM> (coding gene: NM_020661. <NUM>) etc.) and murine AID (e.g., GenBank Accession No. NP_033775. <NUM> (coding gene: NM_009645. <NUM>), etc.), but is not limited thereto.

The tadA may be at least one selected from the group consisting of E. coli tadA (e.g., GenBank Accession No. NP_417054. <NUM>, YP_002408701. <NUM>, YP_002413581. <NUM>, etc.) and is not limited thereto.

By the base conversion (e.g., conversion from cytidine to uridine), single nucleotide substitution can be induced in eukaryotic cells.

The deaminase may be used in the form of a protein, a gene coding therefor (e.g., DNA or mRNA), or a recombinant vector carrying the gene.

As used herein, the target-specific nuclease, also called programmable nuclease, is a generic name for all the nucleases (endonucleases) that can recognize and cleave specific sites on genomic DNA (single strand nick or double strand cleavage).

For example, the target-specific nuclease may be at least one selected from all nucleases that can recognize specific sequences of target genes and have nucleotide cleavage activity to incur indel (insertion and/or deletion) in the target genes.

For example, the target-specific nuclease may include at least one selected from the group consisting of RGENs (RNA-guided engineered nucleases, e.g., Cas protein (i.e., Cas9, etc.), Cpf1, etc.) derived from the CRISPR system, which is a microbial immune system, but is not limited thereto.

The target-specific nuclease may recognize a specific base sequence on a genome in prokaryotic cells and/or animal and plant cells (e.g., eukaryotic cells) including human cells (excluding human embryos to incur a double strand break (DSB). Resulting from the cleavage of double strands of DNA, the double strand break may form a blunt end or a cohesive end. DSB can be effectively repaired by a homologous recombination or non-homologous end-joining (NHEJ) mechanism within cells, during which a desired mutation can be introduced to a target site.

In one embodiment, the target-specific nuclease may be at least one selected from the group consisting of nucleases (e.g., endonucleases) included in the type II and/or type V CRISPR system, such as Cas proteins (e.g., Cas9 protein (CRISPR (Clustered regularly interspaced short palindromic repeats) associated protein <NUM>)), Cpf1 protein (CRISPR from Prevotella and Francisella <NUM>), etc. In this regard, the target-specific nuclease further comprises a target DNA-specific guide RNA for guiding to a target site on a genomic DNA. The guide RNA may be an RNA transcribed in vitro, for example, RNA transcribed from double-stranded oligonucleotides or a plasmid template, but is not limited thereto. The target-specific nuclease may act in a ribonucleoprotein (RNP) form in which the nuclease is associated with guide RNA to form a ribonucleic acid-protein complex (RNA-Guided Engineered Nuclease), in vitro or after transfer to a body (cell).

The Cas protein, which is a main protein component in the CRISPR/Cas system, accounts for activated endonuclease or nickase activity.

The Cas protein or gene information may be obtained from a well-known database such as GenBank at the NCBI (National Center for Biotechnology Information). By way of example, the Cas protein may be at least one selected from the group consisting of:.

The Cpf1 protein, which is an endonuclease in a new CRISPR system distinguished from the CRISPR/Cas system, is small in size relative to Cas9, requires no tracrRNA, and can act with the guidance of single guide RNA. In addition, the Cpf1 protein recognizes a thymine-rich PAM (protospacer-adjacent motif) sequence and cleaves DNA double strands to form a cohesive end (cohesive double-strand break).

By way of example, the Cpf1 protein may be derived from Candidatus sp. , Lachnospira sp. , Butvrivibrio sp. , Peregrinibacteria sp. , Acidominococcus sp. , Porphyromonas sp. , Prevotella sp. , Francisella sp. , Candidatus Methanoplasma, or Eubacterium sp. , e.g., from microbes such as Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA 33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (<NUM>), Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum, Candidatus Paceibacter, Eubacterium eligens, etc., but is not limited thereto.

The target-specific nuclease may be isolated from microbes or may be an artificial or non-naturally occurring enzyme as obtained by recombination or synthesis. For use, the target-specific nuclease may be in the form of an mRNA pre-described or a protein pre-produced in vitro or may be included in a recombinant vector so as to be expressed in target cells or in vivo. In an embodiment, the target-specific nuclease (e.g., Cas9, Cpf1, etc.) may be a recombinant protein made with a recombinant DNA (rDNA). The term "recombinant DNA" means a DNA molecule formed by artificial methods of genetic recombination (such as molecular cloning) to bring together homologous or heterologous genetic materials from multiple sources. For use in producing a target-specific nuclease by expression in a suitable organism (in vivo or in vitro), recombinant DNA may have a nucleotide sequence that is reconstituted with optimal codons for expression in the organism which are selected from codons coding for a protein to be produced.

The target-specific nuclease used herein may be a mutant target-specific nuclease in an altered form. The mutant target-specific nuclease may refer to a target-specific nuclease lacking endonuclease activity of cleaving double strand DNA and may be, for example, at least one selected from among mutant target-specific nucleases mutated to lack endonuclease activity but to retain nickase activity and mutant target-specific nucleases mutated to lack both endonuclease and nickase activities. When the mutant target-specific nuclease has nickase activity, a nick may be introduced to a strand on which base conversion (e.g., conversion of cytidine to uridine) is performed by the deaminase or an opposite strand (e.g., a strand paired to the strand on which base conversion happens) (for example, a nick is introduced between nucleotides at positions <NUM> and <NUM> in the <NUM>'-end direction on the PAM sequence) simultaneously with or successively to the base conversion irrespective of the order. As such, the mutation of the target-specific nuclease (e.g., amino acid substitution, etc.) may occur at least in the catalytically active domain of the nuclease (for example, RuvC catalyst domain for Cas9). In an embodiment, when the target-specific nuclease is a Streptococcus pyogenes-derived Cas9 protein (SwissProt Accession number Q99ZW2(NP_269215. <NUM>); SEQ ID NO: <NUM>), the mutation may be amino acid substitution at least one position selected from the group consisting of a catalytic aspartate residue (e.g., aspartic acid at position <NUM> (D10) for SEQ ID NO: <NUM>, etc.), glutamic acid at position <NUM> (E762), histidine at position <NUM> (H840), asparagine at position <NUM> (N854), asparagine at position <NUM> (N863), and aspartic acid at position <NUM> (D986) on the sequence of SEQ ID NO: <NUM>. A different amino acid to be substituted for the amino acid residues may be alanine, but is not limited thereto.

In another embodiment, the mutant target-specific nuclease may be a mutant that recognizes a PAM sequence different from that recognized by wild-type Cas9 protein. For example, the mutant target-specific nuclease may be a mutant in which at least one, for example, all of the three amino acid residues of aspartic acid at position <NUM> (D1135), arginine at position <NUM> (R1335), and threonine at position <NUM> (T1337) of the Streptococcus pyogenes-derived Cas9 protein are substituted with different amino acids to recognize NGA (N is any residue selected from among A, T, G, and C) different from the PAM sequence (NGG) of wild-type Cas9.

In one embodiment, the mutant target-specific nuclease may have the amino acid sequence (SEQ ID NO: <NUM>) of Streptococcus pyogenes-derived Cas9 protein on which amino acid substitution has been made for:.

As used herein, the term "(a) different amino acids" means (an) amino acids selected from among alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine, lysine, and all variants thereof, exclusive of the amino acid retained at the original mutation positions in wild-type proteins. In one embodiment, the "(a) different amino acids" may be alanine, valine, glutamine, or arginine.

In one embodiment, the mutant target-specific nuclease may be a modified Cas9 protein that lacks endonuclease activity (e.g., but retaining nickase activity or lacking both endonuclease activity and nickase activity) or which recognizes a PAM sequence different from that recognized by wild-type Cas9. For example, the modified Cas9 protein may be a mutant of the Streptococcus pyogenes-derived Cas9 protein (SEQ ID NO: <NUM>), wherein.

By way of example, a mutation at D10 in the Cas9 protein may be D10A mutation (means substitution of A for D at position <NUM> in Cas9 protein; hereinafter, mutations introduced to Cas9 are expressed in the same manner), a mutation at H840 may be H840A, and mutations at D1135, R1335, and T1337 may be D1135V, R1335Q, and T1337R, respectively.

Unless otherwise stated herein, the term "nuclease" refers to "target-specific nuclease", such as Cas9, Cpf1, etc., as described above.

The nuclease may be isolated from microbes or may be an artificial or non-naturally occurring enzyme as obtained by recombination or synthesis. In an embodiment, the nuclease (e.g., Cas9, Cpf1, etc.) may be a recombinant protein made with a recombinant DNA (rDNA). The term "recombinant DNA" means a DNA molecule formed by artificial methods of genetic recombination, such as molecular cloning, to bring together homologous or heterologous genetic materials from multiple organism sources. For use in producing a target-specific nuclease by expression in a suitable organism (in vivo or in vitro), for example, recombinant DNA may have a nucleotide sequence that is reconstituted with optimal codons for expression in the organism which are selected from codons coding for a protein to be produced.

The nuclease may be used in the form of a protein, a nucleic acid molecule coding therefor (e.g., DNA or mRNA), a ribonucleoprotein in which the protein is associated with a guide RNA, a nucleic acid molecule coding for the ribonucleoprotein, or a recombinant vector carrying the nucleic acid molecule.

The deaminase and the nuclease, and/or nucleic acid molecules coding therefor may be in the form that can be translocated into, act within, and/or be expressed within the nucleus.

The deaminase and the nuclease may take a form that is easy to introduce to cells. For example, the deaminase and the nuclease may be linked to a cell penetrating peptide and/or a protein transduction domain. The protein transduction domain may be poly-arginine or an HIV-derived TAT protein, but is not limited thereto.

Because there are various kinds of the cell penetrating peptide or the protein transduction domain in addition to the stated examples, a person skilled in the art may make application of various kinds without limitations to the examples.

In addition, the deaminase and the nuclease, and/or nucleic acid molecules coding therefor may further comprise a nuclear localization signal (NLS) sequence or a nucleic acid sequence coding therefor. Therefore, an expression cassette including a deaminase-encoding nucleic acid molecule and/or a nuclease-encoding nucleic acid molecule may further comprise a regulatory sequence such as a promoter sequence for expressing the deaminase and/or nuclease and optionally an NLS sequence (SEQ ID NO: <NUM>). The NLS sequence is well known in the art.

The deaminase and the nuclease, and/or the nucleic acid coding therefor may be linked to a tag for isolation and/or purification or a nucleic acid coding for the tag. For example, the tag may be selected from the group consisting of small peptide tags, such as His tag, Flag tag, S tag, etc., GST (Glutathione S-transferase) tag, and MBP (Maltose binding protein) tag, but is not limited thereto.

In addition, the base editing composition used in the present disclosure may further comprise a uracil DNA glycosylase inhibitor (UGI) or a coding gene therefor (in the form of a recombinant vector carrying the coding DNA or in the form of mRNA transcribed in vitro). The presence of a uracil DNA glycosylase inhibitor in the base editing composition allows for an increased ratio of the conversion of a specific base by deaminase (i.e., conversion from C to T by cytosine deaminase), compared to the absence thereof. On the other hand, when not further including a uracil DNA glycosylase inhibitor, the base editing composition increases a ratio of substitutions for bases other than a specific base (e.g., substitution of C for T by cytosine deaminase) (that is, substitutions are made on various bases). In one embodiment, the uracil DNA glycosylase inhibitor may be encoded by SEQ ID NO: <NUM>, but is not limited thereto.

As used herein, the term "guide RNA" refers to an RNA that includes a targeting sequence hybridizable with a specific base sequence (target sequence) of a target site in a target gene and functions to associate with a nuclease, such as Cas proteins, Cpf1, etc., and guide the nuclease to a target gene (or target site) in vitro or in vivo (or cells).

The guide RNA may be suitably selected depending on kinds of the nuclease to be complexed therewith and/or origin microorganisms thereof.

For example, the guide RNA may be at least one selected from the group consisting of:.

In detail, the guide RNA may be a dual RNA including CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) or a single guide RNA (sgRNA) including main regions of crRNA and tracrRNA.

The sgRNA may include a region (named "spacer region", "target DNA recognition sequence", "base pairing region", etc.) having a complementary sequence (targeting sequence) to a target sequence in a target gene (target site), and a hairpin structure for binding to a Cas protein. In greater detail, the sgRNA may include a region having a complementary sequence (targeting sequence) to a target sequence in a target gene, a hairpin structure for binding to a Cas protein, and a terminator sequence. These moieties may exist sequentially in the direction from <NUM>' to <NUM>', but is not limited thereto. So long as it includes main regions of crRNA and tracrRNA and a complementary sequence to a target DNA, any guide RNA can be used in the present disclosure.

For editing a target gene, for example, the Cas9 protein requires two guide RNAs, that is, a CRISPR RNA (crRNA) having a nucleotide sequence hybridizable with a target site in the target gene and a trans-activating crRNA (tracrRNA) interacting with the Cas9 protein. In this context, the crRNA and the tracrRNA may be coupled to each other to form a crRNA:tracrRNA duplex or connected to each other via a linker so that the RNAs can be used in the form of a single guide RNA (sgRNA). In one embodiment, when a Streptococcus pyogenes-derived Cas9 protein is used, the sgRNA may form a hairpin structure (stem-loop structure) in which the entirety or a part of the crRNA having a hybridizable nucleotide sequence is connected to the entirety or a part of the tracrRNA including an interacting region with the Cas9 protein via a linker (responsible for the loop structure).

The guide RNA, specially, crRNA or sgRNA, includes a targeting sequence complementary to a target sequence in a target gene and may contain one or more, for example, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> additional nucleotides at an upstream region of crRNA or sgRNA, particularly at the <NUM>' end of sgRNA or the <NUM>' end of crRNA of dual RNA. The additional nucleotide(s) may be guanine(s) (G), but are not limited thereto.

In another embodiment, when the nuclease is Cpf1, the guide RNA may include crRNA and may be appropriately selected, depending on kinds of the Cpf1 protein to be complexed therewith and/or origin microorganisms thereof.

Concrete sequences of the guide RNA may be appropriately selected depending on kinds of the nuclease (Cas9 or Cpf1) (i.e., origin microorganisms thereof) and are an optional matter which could easily be understood by a person skilled in the art.

When a Streptococcus pyogenes-derived Cas9 protein is used as a target-specific nuclease, crRNA may be represented by the following General Formula <NUM>:.

<NUM>'-(Ncas9)l-(GUUUUAGAGCUA)-(Xcas9)m-<NUM>'     (General Formula <NUM>).

In an embodiment, the Xcas9 may include, but is not limited to, UGCUGUUUUG (SEQ ID NO: <NUM>).

In addition, the tracrRNA may be represented by the following General Formula <NUM>:
<IMG>
wherein,.

Further, sgRNA may form a hairpin structure (stem-loop structure) in which a crRNA moiety including the targeting sequence and the essential region thereof and a tracrRNA moiety including the essential region (<NUM> nucleotides) thereof are connected to each other via an oligonucleotide linker (responsible for the loop structure). In greater detail, the sgRNA may have a hairpin structure in which a crRNA moiety including the targeting sequence and an essential region thereof is coupled with the tracrRNA moiety including the essential region thereof to form a double-strand RNA molecule with connection between the <NUM>' end of the crRNA moiety and the <NUM>' end of the tracrRNA moiety via an oligonucleotide linker.

In one embodiment, the sgRNA may be represented by the following General Formula <NUM>:
<IMG>
wherein (Ncas9)l is a targeting sequence defined as in General Formula <NUM>.

The oligonucleotide linker included in the sgRNA may be <NUM>-<NUM> nucleotides long, for example <NUM> nucleotides long in which the nucleotides may be the same or different and are independently selected from the group consisting of A, U, C, and G.

The crRNA or sgRNA may further contain <NUM> to <NUM> guanines (G) at the <NUM>' end thereof (that is, the <NUM>' end of the targeting sequence of crRNA).

The tracrRNA or sgRNA may further comprise a terminator inclusive of <NUM> to <NUM> uracil (U) residues at the <NUM>' end of the essential region (<NUM> nt long) of tracrRNA.

The target sequence for the guide RNA may be about <NUM> to about <NUM> or about <NUM> to about <NUM>, for example, <NUM> consecutive nucleotides adjacent to the <NUM>' end of PAM (Protospacer Adjacent Motif (for S. pyogenes Cas9, <NUM>'-NGG-<NUM>' (N is A, T, G, or C)) on a target DNA.

As used herein, the term "the targeting sequence" of guide RNA hybridizable with the target sequence for the guide RNA refers to a nucleotide sequence having a sequence complementarity of <NUM> % or higher, <NUM> % or higher, <NUM> % or higher, <NUM> % or higher, <NUM> % or higher, <NUM> % or higher, <NUM> % or higher, or <NUM> % to a nucleotide sequence of a complementary strand to a DNA strand on which the target sequence exists (i.e., a DNA strand having a PAM sequence (<NUM>'-NGG-<NUM>' (N is A, T, G, or C))) and thus can complimentarily couple with a nucleotide sequence of the complementary strand.

In the description, a nucleic acid sequence at a target site is represented by that of the strand on which a PAM sequence exists among two DNA strands in a region of a target gene. In this regard, the DNA strand to which the guide RNA couples is complementary to a strand on which a PAM sequence exists. Hence, the targeting sequence included in the guide RNA has the same nucleic acid sequence as a sequence at an on-target site, with the exception that U is employed instead of T due to the RNA property. In other words, a targeting sequence of the guide RNA and a target sequence are represented by the same nucleic acid sequence with the exception that T and U are interchanged, in the description.

The guide RNA may be used in the form of RNA (or may be contained in the composition) or in the form of a plasmid carrying a DNA coding for the RNA (or may be contained in the composition).

As described in the specification, base editing (for example, single nucleotide substitution) can be performed by introducing a deaminase and a target-specific nuclease in the form of, for example, mRNA and RNP to mammalian (e.g., murine) cells through microinjection or electroporation. When such base editing is conducted in non-human mammalian embryos, the embryos can be successfully developed to pups having point mutations induced by the base editing. Taken together, the results indicate that the deaminase and the target-specific nuclease can be used to construct various animal models in which single amino acid substitutions and nonsense mutations are induced.

Hereafter, the present disclosure will be described in detail by examples. The following examples are intended merely to illustrate the invention and are not construed to restrict the invention.

After being isolated by digestion from pCMV-BE3 (Addgene; cat. #<NUM>; <FIG>), rAPOBEC1-XTEN (linker) and UGI (uracil DNA glycosylase inhibitor) were inserted into the pET-nCas9 (D10A)-NLS vector (see<NPL>)) to construct pET-Hisx6-rAPOBEC1-XTEN-nCas9-UGI-NLS (SEQ ID NO: <NUM>; <FIG>) which was then used as a BE3 mRNA template.

Sequences of individual regions in pET-Hisx6-rAPOBEC1-XTEN-nCas9-UGI-NLS (SEQ ID NO: <NUM>) are summarized as follows:.

PCR was performed on the pET-Hisx6-rAPOBEC1-XTEN-nCas9-UGI-NLS vector with the aid of Phusion High-Fidelity DNA Polymerase (Thermo Scientific) in the presence of primers (F: <NUM>'-GGT GAT GTC GGC GAT ATA GG-<NUM>', R: <NUM>'-CCC CAA GGG GTT ATG CTA GT-<NUM>') to prepare the mRNA temperature. From the prepared mRNA template, BE3 mRNA was synthesized using an in vitro RNA transcription kit (mMESSAGE mMACHINE T7 Ultra kit, Ambion), followed by purification with MEGAclear kit (Ambion).

A dystrophin gene Dmd and a tyrosinase gene Tyr targeted guide RNA (sgRNA) having the following nucleotide sequence were synthesized and used in subsequent experiments:
<IMG>.

The sgRNA was constructed by in vitro transcription using T7 RNA polymerase (see<NPL>)).

Rosetta competent cells (EMD Millipore) were transformed with the pET28-Hisx6-rAPOBEC1-XTEN-nCas9(D10A)-UGI-NLS (BE3) expression vector prepared in Reference Example <NUM> and then incubated with <NUM> isopropyl beta-D-<NUM>-thiogalactopyranoside (IPTG) at <NUM> for <NUM> to <NUM> hours to induce expression. Following protein expression, bacterial cells were harvested by centrifugation and the cell pellet was lysed by sonication in a lysis buffer [<NUM> NaH2PO4 (pH <NUM>), <NUM> NaCl, <NUM> imidazole, <NUM>% Triton X-<NUM>, <NUM> PMSF, <NUM> DTT, and <NUM>/ml lysozyme].

The cell lysate thus obtained was subjected to centrifugation at <NUM>,251xg for <NUM> to remove cell debris. The soluble lysate was incubated with Ni-NTA beads (Qiagen) at <NUM> for <NUM> hr. Subsequently, the Ni-NTA beads were washed three times with wash buffer [<NUM> NaH2PO4 (pH <NUM>), <NUM> NaCl, and <NUM> imidazole], followed by eluting BE3 protein with elution buffer [<NUM> Tris-HCl (pH <NUM>), <NUM>-<NUM> NaCl, <NUM>-<NUM>% glycerol, and <NUM> imidazole]. The purified BE3 protein was dialyzed against storage buffer [<NUM> HEPES (pH <NUM>), <NUM> KCl, <NUM> DTT, and <NUM>% glycerol] and concentrated using Ultracell <NUM> cellulose column (Millipore). The purity of the protein was analyzed by SDS-PAGE. SgRNA was prepared by in vitro transcription using T7 RNA polymerase as described in Example <NUM>.

Experiments on mice were conducted after approval by the Institutional Animal Care and Use Committee (IACUC) at Seoul National University. Mice were maintained in a SPF (specific pathogen-free) condition, with a <NUM>/<NUM> hrs light/dark cycle. C57BL/6J and ICR mice were used as an embryo donor and a surrogate mother.

Superovulation, embryo collection, microinjection, and electroporation were performed with reference to "<NPL>)".

For microinjection, a solution containing a complex of BE3 mRNA (<NUM> ng/ul) and sgRNA (<NUM> ng/ul) was diluted in DEPC-treated injection buffer (<NUM> EDTA, <NUM> Tris, pH <NUM>) (see <NPL>)) and then injected into pronuclei of one-cell stage zygote at fertilization with the aid of Nikon ECLIPSE Ti micromanipulator and FemtoJet 4i microinjector (Eppendorf).

For electroporation, a BE3-sgRNA RNP complex was introduced to one-cell mouse embryos by electroporation using a NEPA <NUM> electroporator (NEPA GENE Co. ) including a glass chamber filled with <NUM>µl of opti-MEM (Thermo Fisher Scientific) containing the BE3-sgRNA RNP complex (<NUM>µg/<NUM>µl and <NUM>µg/<NUM>µl, respectively) (see <NPL>)).

After BE3 RNP or mRNA transfer, the embryos were cultured in microdrops of KSOM+AA (Millipore) at <NUM> for <NUM> days under a humidified condition of <NUM>% CO2. Two-cell stage embryos were transplanted to the oviduct of a <NUM>-dpc pseudo pregnant surrogate mother.

The procedure is summarized in the following diagram:
<IMG>.

For PCR genotyping, genomic DNA was extracted from the blastula stage embryo obtained from the embryo transplanted to the oviduct in Reference Example <NUM> or from an ear clip of the newborn pups and subjected to targeted deep sequencing and Sanger sequencing.

In this Example, nucleotide sequences were analyzed using targeted deep sequencing as follows.

Target sites or off-target sites were amplified from the genomic DNA extracted in Reference Example <NUM> with the aid of Phusion polymerase (Thermo Fisher Scientific). Paired-end sequencing of the PCR amplicons was performed using Illumina MiSeq (LAS, Inc. (South Korea) commissioned to perform). Primers used in the amplification of off-target sites are given in Tables <NUM> and <NUM>, blow.

Tibialis anterior (TA) muscle sections resected from the mouse were immunostained with a laminin or dystrophin antibody. Laminin was detected using a <NUM>:<NUM> dilution of a rabbit polyclonal antibody (abcam, ab11575) and a <NUM>:<NUM> dilution of an Alexa Fluor <NUM> anti-rabbit secondary antibody (Thermo Fisher Scientific) sequentially. For dystrophin detection, a <NUM>:<NUM> dilution of rabbit polyclonal antibody (abcam, ab15277) and a <NUM>:<NUM> dilution of an Alexa Fluor <NUM> anti-rabbit secondary antibody (Thermo Fisher Scientific) were used sequentially. The immunofluorescently stained sections were observed with a Leica DMI4000 B fluorescence microscope.

As described in Examples <NUM>-<NUM>, Base Editor <NUM> (BE3) (rAPOBEC1-nCas9-UGI) was introduced to mouse embryonic cells by microinjection to induce a point mutation in each of the dystrophin-encoding gene Dmd and the tyrosinase-encoding gene Tyr.

As shown in <FIG> (Dmd) and 2a (Tyr), the generation of a stop codon would be predicted by single base substitution (C→T) at a target site (underlined sequence sites of upper sequences in <FIG> and <FIG>) in each gene (in <FIG> and <FIG>, single base substitutions occurred on lower nucleotide sequences, with substituted (C→T) bases appearing red).

<FIG> and <FIG> show summaries of procedures of target-specific single base substitution (microinjection or electroporation) and results thereof. As shown in <FIG> and <FIG>, embryonic mutations were observed at target sites in Dmd and Tyr genes at frequencies of <NUM> % (<NUM> of <NUM> for Dmd) and <NUM> % (<NUM> of <NUM> for Tyr), respectively.

In addition, nucleotide sequences of target sites in mouse embryos in which mutations had been induced by microinjecting BE3 mRNA and sgRNA were identified by targeted deep sequencing. In greater detail, target-specific mutation was induced by microinjecting mouse BE3 (rAPOBEC1-nCas9-UGI) mRNA and sgRNA into mouse embryos. For this, BE3-encoding mRNA and sgRNA were microinjected to mouse zygotes, and then nucleotide sequences of target sites in the target genes (Dmd and Tyr) in the resulting blastocysts were aligned.

(Wt, wild-type; the target sequence is underlined; the PAM sequence (NGG) is shown in bold; substituted bases are in bold and underlined; the column on the right indicate frequencies (%) of mutant (base substituted) alleles and '-' stands for absence of nucleotides at corresponding positions (deletion); numerals on the left are mutated mouse embryonic cell numbers).

As is understood from the aligned sequences, C→T base substitution is a predominant mutation pattern at both the target sites in the two genes (Dmd and Tyr).

After microinjection of BE3 mRNA and Dmd targeted sgRNA thereinto, mouse embryos were transplanted to the oviduct of foster surrogate mothers (see Example <NUM>) to give mutant newborn pups having point mutation on the Dmd gene thereof (F0).

<FIG> and <FIG> show analysis results of nucleotide sequences of the target site in the target gene (Dmd) of newborn pups developed after BE3 (rAPOBEC1-nCas9-UGI)-encoding mRNA and sgRNA hybridizable with the nucleotide sequence of the target site in the Dmd gene have been injected into moue zygotes. As can be seen in <FIG>, when point mutation was induced in the Dmd gene, five (D102, D103, D107, D108, and D109) among a total of nine mice had mutation at the target site in the Dmd gene. Of the five mutant mice, three subjects (D102, D103, and D108) were found to have one or two mutant allele genes and lack wild-type allelomorphic characteristics. The other two mutant mice (D107 and D109) retained wild-type allele genes in a mosaic pattern at a frequency of <NUM> %. In addition, as can be seen <FIG> and <FIG>, the mutant mouse D109 exhibited <NUM>-base pair (bp) deletion other than point mutation, demonstrating that the Cas9 nickase included in BE3 retains the activity of inducing indels at the target site.

<FIG> shows Sanger sequencing chromatograms of the target site in the target gene of wild-type mouse and Dmd mutant mouse D108. As shown in <FIG>, the mutant F0 mouse D108, which lacks a wild-type allele gene, had an early stop codon (TAG) introduced by single base substitution (C→T) to the Dmd gene thereof.

<FIG> shows images of immunofluorescent stained TA muscle sections from the wild-type mouse and the Dmd mutant mouse D108 (see Example <NUM>), exhibiting that dystrophin was nearly not expressed in the muscle of the mutant subject (D108). The result implies that the Dmd gene was successfully knocked down by the injection (microinjection of BE3 mRNA and Dmd targeted sgRNA.

BE3 ribonucleproteins (RNPs), prepared in Example <NUM>, including a mixture (rAPOBEC1-nCas9(D10A)-UGI RNP) of the recombinant BE3 protein and the in-vitro transcribed sgRNA were transferred to mouse embryos by electroporation (see Example <NUM>). Four days after electroporation, nucleotide sequences of target sites in the target genes (Dmd and Tyr) of the mouse embryos were analyzed and the results are given as follows:
<IMG>.

(Wt, wild-type; the target sequence is underlined; the PAM sequence (NGG) is shown in bold; substituted bases are in bold and underlined; the column on the right indicate frequencies (%) of mutant (base substituted) alleles and '-' stands for absence of nucleotides at corresponding positions (deletion); numerals on the left are mutated mouse numbers).

As understood from the results and <FIG> and <FIG>, the electroporation induced mutations at the Dmd and Tyr target sites in the blastocyst embryos at frequencies of <NUM> % (<NUM> of <NUM> for Dmd) and <NUM> % (<NUM> of <NUM> for Tyr).

After electroporation of BE3 and Tyr targeted sgRNA thereinto, mouse embryos were transplanted to the oviduct of surrogate mothers (see Example <NUM>) to give mice having point mutation on the Tyr gene thereof (F0).

<FIG> shows alignment of nucleotide sequences at the target site in Tyr genes of the mutant newborn pups thus obtained. As shown in <FIG>, various mutations were induced at the target site in the Tyr gene of all of the seven mutant newborn pups (T110, T111, T112, T113, T114, T117, and T118).

<FIG> shows Sanger sequencing chromatograms of the target site in the target gene of wild-type mouse and mutant newborn pups (T113 and T114). As seen, the mutant newborn pups had a stop codon (TAG) introduced by single base substitution (C→T) to the target site.

<FIG> shows phenotypes of the eyes of the mutant newborn pups, exhibiting ocular albinism in the mutant mice (T113 and T114). The result implies that the Tyr gene was successfully knocked down by the introduction (electroporation) of RNP of BE3 mRNA and Tyr targeted sgRNA.

In order to assay off-target effects of BE3, potential off-target sites having up to <NUM>-nucleotide mismatches were found in the moue genome by using the Cas-OFFinder (http://www. net/cas-offinder/) and genomic DNA isolated from the mutant newborn pups were analyzed using targeted deep sequencing.

sgRNA sequence and primer sequences used in targeted deep sequencing are summarized in Table <NUM>, below.

The off-target sites identified in the mouse genome and the targeted deep sequencing results are given in Tables <NUM> (Dmd) and <NUM> (Tyr) and <FIG> (Dmd) and <NUM> (Tyr).

(In Tables <NUM> and <NUM>, mismatched nucleotides in off-target sites with the on-target sequence are represented in lower cases: NGG at the <NUM>' end accounts for the PAM sequence).

Claim 1:
A base editing method for a non-human mammalian embryonic cell, the method comprising:
directly injecting <NUM>) a ribonucleoprotein complex or mixture, the ribonucleoprotein complex or mixture comprising a cytidine deaminase, an RNA-guided nuclease, and a guide RNA, or <NUM>) a mixture of a cytidine deaminase-encoding mRNA, an RNA-guided nuclease-encoding mRNA, and a guide RNA,
into the non-human mammalian embryonic cell without using a recombinant vector.