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WO2025202473A1 - Déaminase d'acide nucleique, éditeur de bases et utilisations associées - Google Patents

Déaminase d'acide nucleique, éditeur de bases et utilisations associées

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Publication number
WO2025202473A1
WO2025202473A1 PCT/EP2025/058591 EP2025058591W WO2025202473A1 WO 2025202473 A1 WO2025202473 A1 WO 2025202473A1 EP 2025058591 W EP2025058591 W EP 2025058591W WO 2025202473 A1 WO2025202473 A1 WO 2025202473A1
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nucleic acid
tada
sequence
deaminase
seq
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Inventor
Robin Léo Philippe LOESCH
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Revvity Discovery Ltd
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Revvity Discovery Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04004—Adenosine deaminase (3.5.4.4)

Definitions

  • Base editing is an approach to genome editing that enables the direct conversion of one nucleobase into another in a programmable manner.
  • Base editing does not require doublestranded nucleic acid backbone (e.g., DNA) cleavage, a donortemplate or relying on cellular homology directed repair (HDR). Accordingly, base editing may be a safer alternative to conventional genome editing approaches (e.g., RNA-programmable CRISPR-associated (Cas) nucleases) by minimizing the risks to cells created by double-stranded breaks (DSBs), such as the formation of DSB-associated byproducts.
  • DSBs double-stranded breaks
  • DNA base editors may comprise fusions between a catalytically impaired Cas nuclease (e.g., Cas nickase/nCas or a dead Cas/dCas) and a base-modification enzyme that operates on single-stranded DNA (ssDNA).
  • a catalytically impaired Cas nuclease e.g., Cas nickase/nCas or a dead Cas/dCas
  • ssDNA single-stranded DNA
  • base-modification enzyme e.g., a nucleic acid deaminase
  • Cytidine base editors rely on naturally occurring enzymes to convert a C-G base pair into a T-A base pair (C-to-T transition mutation).
  • Adenine base editors use a modified version of a transfer RNA adenosine deaminase enzyme to convert an A-T base pair to a G-C base pair (A-to-G transition mutation).
  • an ABE catalyses deamination of an adenosine within the ssDNA yielding inosine, which in the context of transcription or replication exhibits the base-pairing preference of guanosine in the active site of a polymerase.
  • ecTadA Escherichia coliTad
  • ssDNA single-stranded DNA
  • ABEs adenosine base editors
  • a directed protein evolution strategy transformed a protein with no ability to deaminate adenine at target loci in DNA into forms that edit DNA, identifying an ABE protein that comprises as many as 14 mutations to achieve a good base editing efficiency.
  • adenosine deaminases Available approaches for base editing using adenosine deaminases are currently suffering from various limitations including: (i) a lack of variety of adenosine deaminases acting on DNA, as the ones identified so far are fused to nCas9 in a single polypeptide chain to generate sufficient editing rates, (ii) low total DNA base editing activity, (iii) compatibility of the TadA deaminase with sequence-targeting proteins, (iv) base editing window preference (e.g., at canonical positions A5-A7 in a protospacer compared with non- canonical positions A3, A4, A8-A10), (v) broad base editing window, (vi) presence of off- target modifications, (vii) variable efficiency depending on the -1 nucleotide, etc. Moreover, there remains a need in the field for more specific, controlled, and safer methods of base editing nucleic acids in cells, in particular, in human and animal cells.
  • the present disclosure also provides methods and uses which employ the disclosed nucleic acid deaminase orthe disclosed base editor for base editing in a nucleic acid molecule including methods for modifying a nucleic acid molecule (e.g., to obtain a genetically engineered isolated cell); methods for target site-specific modification of a nucleic acid molecule; methods of preventing or treating subjects having or at risk of developing a disease, disorder or condition associated with a point mutation; and uses as a basic research tool and/or a screening tool.
  • the present disclosure is defined in the corresponding independent claims.
  • the present disclosure provides a nucleic acid deaminase capable of deaminating a deoxyadenosine (dA) in a nucleic acid molecule (e.g., DNA).
  • the nucleic acid deaminase comprises an amino acid sequence of a wild-type TadA, wherein one or more amino acid residue(s) of the wild-type TadA are deleted, and wherein the one or more deleted amino acid residue(s) are located within a region of the wild-type TadA contacting the nucleic acid molecule (e.g.,
  • TadA deletion variants Evolved TadA enzymes that enable base conversion of A-T to G-C base pairs in DNA (A-to- G transition) were centered around amino acid substitutions (existing TadA substitution variants), whereas deletions of amino acids remained overlooked.
  • the inventors have found that the TadA deletion variants according to the present disclosure achieve a base editing activity (e.g., DNA editing activity as defined by percentage of A-to-G transition, as measured by sequencing) that is equal to, or higher compared to a TadA substitution variant that has a substitution at the corresponding deleted amino acid residue. Accordingly, the disclosed TadA deletion variants are able to deaminate new substrates (e.g., DNA substrates as compared to RNA substrates for wild-type TadA).
  • the nucleic acid deaminase of the present disclosure achieves a similar base editing efficiency (transition of A-to-G) with only one, two or three amino acid deletions.
  • the inventors have also found that the previously reported substitution D108N of ecTadA is not essential for creating a nucleic acid deaminase with detectable DNA editing activity.
  • a "free" nucleic acid deaminase according to the present disclosure can deaminate any exposed dA in a nucleic acid molecule (e.g., a DNA).
  • a nucleic acid molecule e.g., a DNA
  • the present disclosure provides a base editor comprising (i) an effector protein comprising at least one nucleic acid deaminase according to the present disclosure, (ii) a sequence-targeting protein, and (iii) an RNA-ligand binding complex.
  • the base editor of the present disclosure is specific for a target site in a nucleic acid molecule, wherein the sequence-targeting protein (ii) and the RNA-ligand binding complex (iii) are capable of recruiting the effector protein (i) to the target site (e.g., a protospacer) in the nucleic acid molecule.
  • the base editor of the present disclosure showed desirable base editing window preference (e.g., at canonical positions A5-A7, All in a protospacer compared with non- canonical positions A3, A4, A8-A10) and a narrow base editing window (e.g., at a single dA).
  • the present inventors showed that the base editor of the present disclosure created precise A-to-G transitions with equal or fewer bystander edits compared to a base editor comprising a TadA substitution variant that has a substitution at the corresponding deleted amino acid residue (existing adenosine base editor, ABE).
  • the disclosed base editor is not limited to a specific sequence motif at the target site (in contrast, a wild-type ecTadA has a strict UACG sequence motif requirement in RNA; see, e.g., Fig. 1 of Kim et al. Biochemistry, Vol. 45, No. 20, 2006, doi: 10.1021/bi0522394).
  • adenosine base editor include a fusion protein containing a heterodimer of a wild-type (WT) TadA monomer that plays a structural role during base editing and a TadA substitution variant monomer that catalyzes dA deamination.
  • WT wild-type
  • TadA substitution variant monomer that catalyzes dA deamination.
  • a base editor comprising a monomer of the disclosed TadA deletion variant was capable of maintaining or improving DNA base editing levels compared to a base editor comprising a heterodimer of a WT TadA monomer and the disclosed TadA deletion variant or a homodimer of two disclosed TadA deletion variants.
  • a base editor comprising (i) an effector protein comprising at least one nucleic acid deaminase according to the present disclosure and at least one ligand capable of binding to a ligand binding moiety (e.g., an RNA binding domain such as MCP), (ii) a sequence-targeting protein, and (iii) an RNA-ligand binding complex comprising at least one ligand binding moiety (e.g., an RNA motif selected from a single MS2 RNA motif, Qbeta RNA motif, boxB RNA motif, Csy4 RNA motif or PP7 RNA motif) had an equal or improved base editing performance (also referred to as "aptamer-recruitment- dependent base editing system”) compared to existing adenosine base editors based on a fusion between the deaminase and the sequence-targeting protein.
  • a ligand binding moiety e.g., an RNA binding domain such as MCP
  • a sequence-targeting protein
  • the present inventors also found that the disclosed base editor enables concurrent adenine and cytosine editing (a dual-function base editor).
  • a first base editor comprising at least one nucleic acid deaminase according to the present disclosure may be multiplexed with at least one further base editor.
  • the inventors showed that multiplexing a first base editor comprising a nucleic acid deaminase according to the present disclosure and a second base editor comprising a further protein capable of site-specific deamination (e.g., a cytidine deaminase) yielded combined DNA editing of A-to-G and C-to-T at the target site.
  • site-specific deamination e.g., a cytidine deaminase
  • the disclosed base editor is not limited to a specific sequence motif at the target site
  • the present disclosure provides methods and uses which employ the disclosed nucleic acid deaminase or the disclosed base editor for base editing in a nucleic acid molecule.
  • the methods disclosed herein can be used to modify specific target sites contained in nucleic acid molecules of eukaryotic and prokaryotic cells.
  • the methods can further be used to modify specific target sites contained in nucleic acid molecules within organelles (e.g., chloroplasts and/or mitochondria).
  • the methods can further be used to generate a cell (e.g., a genetically engineered isolated cell).
  • the disclosed nucleic acid deaminase and the disclosed base editor are also useful as a basic research tool (e.g., to identify, test, and/or manipulate target site(s) in a nucleic acid molecule), and/or as a screening tool (e.g., diagnostic screens).
  • the present disclosure provides the disclosed nucleic acid deaminase or the disclosed base editor for medical applications to prevent or treat diseases, disorders or conditions that are associated or caused by one or more point mutation(s) that may be corrected by deaminase-mediated base editing (e.g., as a medicament, for treating or preventing a genetic disease, or for correcting a pathogenic point mutation or a loss-of- function mutation).
  • deaminase-mediated base editing e.g., as a medicament, for treating or preventing a genetic disease, or for correcting a pathogenic point mutation or a loss-of- function mutation.
  • Precise and efficient base editing is critical for therapeutic applications (e.g., in gene therapy), as any bystander or off-target edit may result in undesired mutations in an (on- or off-) target site.
  • a nucleic acid deaminase capable of deaminating a deoxyadenosine (dA) in a nucleic acid molecule wherein the nucleic acid deaminase comprises an amino acid sequence of a wild-type TadA, wherein one or more amino acid residue(s) of the wildtype TadA are deleted, and wherein the one or more deleted amino acid residue(s) are located within a region of the wild-type TadA contacting the nucleic acid molecule.
  • nucleic acid deaminase according to item 1, wherein the deoxyadenosine (dA) is contained in a double-stranded or single-stranded DNA region of the nucleic acid molecule.
  • the nucleic acid deaminase according to item 2 wherein the deoxyadenosine (dA) is contained in a single-stranded DNA region of the nucleic acid molecule; optionally wherein the single-stranded DNA region is part of an R-loop.
  • nucleic acid deaminase according to any one of items 1 to 4, wherein the region of the wild-type TadA contacting the nucleic acid molecule comprises amino acid residues 105 to 130 of E.coli TadA shown in SEQ ID NO: 1 or corresponding amino acid residues in a bacterial TadA shown in one of SEQ ID NOs: 2 to 8.
  • the nucleic acid deaminase according to any one of items 1 to 5 wherein the region of the wild-type TadA contacting the nucleic acid molecule consists of amino acid residues 105 to 130 of E.coli TadA shown in SEQ ID NO: 1 or corresponding amino acid residues in a bacterial TadA shown in one of SEQ ID NOs: 2 to 8.
  • nucleic acid deaminase according to any one of items 1 to 6, wherein the region of the wild-type TadA contacting the nucleic acid molecule comprises amino acid residues 105 to 110 of E.coli TadA shown in SEQ ID NO: 1 or corresponding amino acid residues in a bacterial TadA shown in one of SEQ ID NOs: 2 to 8.
  • the nucleic acid deaminase according to any one of items 1 to 7 wherein the region of the wild-type TadA contacting the nucleic acid molecule consists of amino acid residues 105 to 110 of E.coli TadA shown in SEQ ID NO: 1 or corresponding amino acid residues in a bacterial TadA shown in one of SEQ ID NOs: 2 to 8.
  • a base editor comprising (i) an effector protein comprising at least one nucleic acid deaminase according to any one of items 1 to 17.
  • the effector protein comprises one or two nucleic acid deaminase(s) according to any one of items 1 to 17.
  • the at least one nucleic acid deaminase comprises an amino acid sequence derived from any one of SEQ ID NOs: 9 to 15.
  • the effector protein further comprises at least one ligand capable of binding to a ligand binding moiety.
  • the base editor according to any one of items 18 to 25, wherein the base editor further comprises (ii) a sequence-targeting protein.
  • sequence-targeting protein is a nuclease comprising at least one catalytically inactive nuclease domain.
  • sequencetargeting protein is a CRISPR-Cas protein with at least one catalytically inactive nuclease domain (nickase) or a nuclease-dead CRISPR-Cas protein.
  • the ligand binding moiety is an RNA motif selected from a single MS2 phage operator stem-loop (MS2) RNA motif, Qbeta RNA motif, boxB RNA motif, telomerase Ku binding motif, telomerase Sm7 binding motif, SfMu phage Com stem-loop, Csy4 RNA motif and PP7 phage operator stem-loop RNA motif.
  • MS2 phage operator stem-loop MS2 phage operator stem-loop
  • Qbeta RNA motif boxB RNA motif
  • telomerase Ku binding motif telomerase Sm7 binding motif
  • SfMu phage Com stem-loop SfMu phage Com stem-loop
  • Csy4 RNA motif Csy4 RNA motif
  • PP7 phage operator stem-loop RNA motif PP7 phage operator stem-loop RNA motif.
  • the effector protein further comprises at least one ligand capable of binding to a ligand binding moiety, wherein the ligand is an RNA binding domain, and wherein in (iii) the ligand binding moiety is an RNA motif.
  • RNA moiety capable of binding to the sequence-targeting protein comprises a tracrRNA or scoutRNA
  • the RNA moiety capable of binding to a target site comprises a crRNA
  • an effector protein comprising at least one nucleic acid deaminase according to any one of items 1 to 17 and at least one ligand capable of binding to a ligand binding moiety
  • RNA-ligand binding complex comprising at least one ligand binding moiety; and wherein the at least one ligand of the effector protein (i) binds to the ligand binding moiety of the RNA-ligand binding complex (iii).
  • RNA-ligand binding complex comprising (a) an RNA moiety capable of binding to the sequence-targeting protein, (b) an RNA moiety capable of binding to a target site and (c) at least one ligand binding moiety; and wherein the at least one ligand of the effector protein (i) binds to the ligand binding moiety of the RNA-ligand binding complex (iii).
  • the base editor according to any one of items 18 to 31 and 34 to 45, wherein the base editor comprises:
  • an effector protein comprising one nucleic acid deaminase having an amino acid sequence as set forth in any one of SEQ ID NOs: 9 to 15 and at least one ligand capable of binding to a ligand binding moiety
  • a sequence-targeting protein comprising a CRISPR-Cas protein with at least one catalytically inactive nuclease domain (nickase) or a nuclease-dead CRISPR-Cas protein, and
  • RNA-ligand binding complex comprising (a) an RNA moiety capable of binding to the sequence-targeting protein, (b) an RNA moiety capable of binding to a target site and (c) at least one ligand binding moiety; and wherein the at least one ligand of the effector protein (i) binds to the ligand binding moiety of the RNA-ligand binding complex (iii).
  • the base editor according to any one of items 18 to 46, wherein the deoxyadenosine (dA) is in a target site in the nucleic acid molecule.
  • the target site comprises a deoxyadenosine (dA) and a protospacer.
  • PAM protospacer adjacent motif
  • the base editor according to any one of items 18 to 50, wherein the base editor is a deoxyadenosine (dA) base editor.
  • dA deoxyadenosine
  • nucleic acid molecule is a DNA selected from the group consisting of genomic DNA, nuclear DNA, chromosomal DNA, organellar DNA, exogenous DNA, viral DNA and a stably maintained plasmid.
  • a cell comprising the nucleic acid deaminase according to any one of items 1 to 17.
  • a cell comprising the base editor according to any one of items 18 to 52, wherein the cell is obtained from a prokaryotic cell or a eukaryotic cell.
  • a cell comprising the isolated nucleic acid, the expression construct, or the vector according to any one of items 53 to 55.
  • kit of parts comprising part (i), wherein part (i) comprises an effector protein comprising at least one nucleic acid deaminase according to any one of items 1 to 17, and part (ii), wherein part (ii) comprises a sequence-targeting protein.
  • kit of parts according to item 62 further comprising part (iii), wherein part (iii) comprises an RNA-ligand binding complex.
  • kit of parts according to item 63 comprising parts (i), (ii) and (iii), wherein in part (i) the effector protein further comprises at least one ligand capable of binding to a ligand binding moiety, wherein in part (ii) the sequence-targeting protein is a CRISPR-Cas protein, and wherein in part (iii) the RNA-ligand binding complex comprises at least one ligand binding moiety; and wherein the at least one ligand of part (i) is capable of binding to the at least one ligand binding moiety of part (iii).
  • kit of parts according to any one of items 62 to 64, wherein the parts (i), (ii), and/or (iii) are encoded on one or more expression construct(s).
  • a method for modifying a nucleic acid molecule comprising contacting the nucleic acid molecule with at least one nucleic acid deaminase according to any one of items 1 to 17.
  • nucleic acid molecule comprises at least one deoxyadenosine (dA).
  • a method for target site-specific modification of a nucleic acid molecule comprising contacting the nucleic acid molecule with a base editor according to any one of items 18 to 52 at a target site.
  • the base editor comprises (i) an effector protein, (ii) a sequence-targeting protein and (iii) an RNA-ligand binding complex, and wherein in (iii) the RNA-ligand binding complex comprises an RNA moiety capable of binding to the sequence-targeting protein and an RNA moiety capable of binding to the target site.
  • the method for target site-specific modification of a nucleic acid molecule according to any one of items 71 to 74 comprising a first step of contacting the nucleic acid molecule with a first base editor according to any one of items 18 to 52 at a first target site and a simultaneous or subsequent second step of contacting the nucleic acid molecule with a second base editor at the first target site or at a second target site.
  • the method for target site-specific modification of a nucleic acid molecule according to item 75 wherein the first target site and the second target site are on the same nucleic acid molecule or different nucleic acid molecules.
  • 77 The method for target site-specific modification of a nucleic acid molecule according to any one of items 72 to 76, wherein the deoxyadenosine (dA) is in a target site in the nucleic acid molecule.
  • RNA-ligand binding complex (iii) is delivered into a cell.
  • nucleic acid molecule is a DNA molecule.
  • nucleic acid molecule is a DNA selected from the group consisting of genomic DNA, nuclear DNA, chromosomal DNA, organellar DNA, exogenous DNA, viral DNA and a stably maintained plasmid.
  • Figure 1 is an overview of the designs of the Escherichia coli transfer RNA adenosine deaminase enzyme deletion (ecTadAA) variants generated. It shows amino acid residues 105 to 130 ("P4-P5 loop segment") of the wild-type ecTadA deaminase sequence (SEQ ID NO: 1), referred here as WT, which are located in the
  • 3D) protospacer sequences using an aptamer-recruitment-dependent base editing system comprising the different ecTadAA variants C-terminally fused to MCP: Dl-MCP, D2-MCP, D3-MCP, D4-MCP, D5-MCP, D6-MCP and D7-MCP, the SpCas9_D10A nuclease and a gRNA containing an MS2 aptamer in 3' position.
  • the MS2 aptamer will recruit the ecTadAA variants fused to MCP protein.
  • the values shown represent the degree of A-to-G transition (in percentage) for each nucleotide across the corresponding protospacer.
  • NT represents the non-transfected control.
  • G-to-G events were excluded from the analysis, shown as "-" (not applicable).
  • Polypeptide “peptide”, and “protein”, as used herein, are used interchangeably herein and refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • the term includes fusion proteins including fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residue, immunologically tagged proteins, and the like.
  • Fusion protein refers to a non-naturally occurring fusion which contain one or more protein(s).
  • the linker is 1-100 amino acids in length, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.
  • Recombinant refers to a nucleic acid, a protein, a cell, or an organism that is artificially produced (e.g., formed by laboratory methods).
  • Recombinant nucleic acid refers to a nucleic acid that is the product of various combinations of cloning, restriction, and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from nucleic acids found in natural systems.
  • Recombinant polypeptide refers to a polypeptide that is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of amino acid(s).
  • Nucleic acid and polynucleotide are used interchangeably, and refer to biopolymers containing nucleotides.
  • Nucleic acid molecules refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • nucleic acid molecule refers to biopolymers containing nucleotides (see, e.g., "nucleic acid”).
  • nucleic acid deaminase refers to biopolymers containing nucleotides (see, e.g., "nucleic acid”).
  • the nucleic acid molecule comprises at least one base as a substrate for a nucleic acid deaminase (e.g., a deoxyadenosine, dA).
  • dA deoxyadenosine
  • the nucleic acid molecule comprises a "DNA region" comprising the at least one dA.
  • isolated refers to a nucleic acid, a protein, a cell, or an organism that is in an environment different from that in which the nucleic acid, the protein, the cell, or the organism naturally occurs.
  • isolated nucleic acid can encompass naturally occurring as well as artificial (e.g., chemically or enzymatically modified) parts or building blocks.
  • isolated cell can encompass a cell that is substantially separated from other cells of a tissue.
  • regulatory element refers to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate expression of a (non) coding sequence and/or production of the encoded polypeptide in a host cell.
  • Vector refers to a vehicle (e.g., a molecule or complex) to transfer genetic material into a cell for the purpose of the expression and/or propagation of the genetic material, or to be used in the construction of other genetic material.
  • a vector is a plasmid (e.g., a stably maintained plasmid), a virus or bacteriophage, a cosmid or an artificial chromosome.
  • a vector refers to a nucleic acid molecule harboring at least one origin of replication, a multiple cloning site (MCS) and one or more selection marker(s).
  • MCS multiple cloning site
  • a vector can be introduced into cells and organisms to express RNA transcripts, proteins, and peptides, and may be termed an "expression vector".
  • Cell refers to an in vivo or in vitro cell, such as an in vivo or in vitro eukaryotic cell, prokaryotic cell, or a cell obtained from a multicellular organism (e.g., a cell line) used as recipients for a nucleic acid (e.g., an expression vector), and/or a polypeptide (e.g., a ribonucleoprotein, "RNP" complex).
  • a recombinant host cell is a host cell which has been introduced a nucleic acid (e.g., an expression vector), and/or a polypeptide (e.g., RNP complex).
  • the cell is an in vitro cell that is not the human body, at the various stages of its formation and development.
  • Genetically modified cell or “genetically engineered cell”, as used herein, are used interchangeably and refer to an in vivo or in vitro cell, such as an in vivo or in vitro eukaryotic cell, prokaryotic cell, or a cell obtained from a multicellular organism (e.g., a cell line) which has been modified in its nucleic acid, such as its extrachromosomal nucleic acid or its genomic nucleic acid or genomic nucleic acid on a chromosome.
  • the genetically engineered isolated cell can be modified by the method of the present disclosure in its DNA genome or in an extrachromosomal DNA or in genomic DNA on a chromosome.
  • Transformation refers to a transient or a permanent genetic change induced in a cell following introduction of a vector into the cell (e.g., a nucleic acid exogenous to the cell). Suitable methods for genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like. The choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (e.g., in vitro, ex vivo, or in vivo).
  • Sequence identity as used herein, with respect to a nucleic acid sequence or a protein sequence is defined as the percentage of nucleotides or amino acid residues in a candidate sequence that are identical with the nucleotides or amino acid residues in the specific (parental) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide or amino acid sequence identity can be achieved in various ways that are within the skill in the art. For instance, using publicly available computer software such as BLAST (available at ncbi.nlm.nih.gov/BLAST (Altschul et al J Mol Biol.
  • Substantially complementary refers to a degree of complementarity that is at least 60%, 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%, or at least 99%, over a region of, for example, 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 consecutive nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
  • Wild-type sequence and WT sequence and WT are used interchangeably, and refer to an amino acid sequence or a nucleotide sequence that can be used as template for subsequent reactions or modifications.
  • the wild-type sequence may include a nucleic acid sequence (such as DNA or RNA or combinations thereof) or an amino acid sequence or may be composed of different chemical entities.
  • the sequence initially provided would be regarded as wild-type sequence in view of downstream processes based thereon, irrespective of whether the sequence itself is a natural (i.e., is found in nature, including allelic variations and has not been intentionally modified) or a modified sequence (e.g., the sequence was modified with regard to another wild-type sequence or is completely artificial).
  • a nucleic acid wild-type sequence may be obtained by PCR amplification of a corresponding template region or may be synthesized de novo based on assembly of synthetic oligonucleotides.
  • Wild-type TadA refers to the wild-type (WT) TadA enzyme which comprises a five-stranded beta-sheet core, with five alpha-helices wrapped around to form the active site.
  • wild-type TadA displays a loop region that joins the beta4 and beta5 strands ("loop between
  • 35 loop comprises or consists of residue numbers 118 to 142 (24 amino acids) (Fig. 2 of Kim et al. Biochemistry, Vol. 45, No. 20, 2006, doi: 10.1021/bi0522394; Rallapalli et al. 2020, doi: 10.1126/sciadv.aaz2309).
  • 35 loop segment refers to amino acid residues from 105 to 130 of the wild-type ecTadA or a corresponding region in other wild-type TadA proteins from other organisms.
  • a region of the wild-type TadA contacting the nucleic acid molecule refers to a region contacting the target nucleic acid molecule or a corresponding region in other wild-type TadA proteins.
  • Nucleic acid deaminase refers to enzymes involved in purine metabolism or pyrimidine metabolism.
  • a "nucleic acid deaminase capable of deaminating a deoxyadenosine (dA) in a nucleic acid molecule” as used herein refers to a deaminase enzyme that acts on deoxyadenosine (also referred to as “adenosine deaminase”) and deaminates the deoxyadenosine to convert it into an inosine, which base pairs like guanine (G) in the context of DNA.
  • Nucleic acid deaminase variant or “TadA variant”, as used herein, refers to a polynucleotide or polypeptide of a nucleic acid deaminase which has a sequence substantially similar to a reference WT polynucleotide or polypeptide.
  • the "nucleic acid deaminase variant” or “TadA variant” comprises at least one amino acid residue difference (e.g., a deletion, insertion or a substitution) compared to a wild-type nucleic acid deaminase.
  • “Mutation” refers to a substitution of a residue within a sequence with another residue (e.g., a nucleic acid or amino acid sequence), a deletion of one or more residues within a sequence (e.g., a nucleic acid or amino acid sequence), or an insertion of one or more residues within a sequence (e.g., a nucleic acid or amino acid sequence).
  • “mutation of an amino acid residue” means that a specific amino acid residue in an amino acid sequence of a protein is substituted, deleted or inserted with an amino acid residue different from an amino acid residue in a corresponding wild-type amino acid sequence.
  • the determination of the presence or absence of the mutation of the amino acid residue described above may be performed by known methods. Methods to obtain a mutated sequence by chemical, enzymatic or other means are known in the art.
  • One or more mutation(s) refers to a one or more mutation are selected from any one of the mutations which have an effect on the editing efficiency, nucleotide context preference and/or off targets.
  • mutation may be selected from mutations described in Zhou, C. et al. Nature 571, 275-278 (2019), doi: 10.1038/s41586- 019-1314-0; Li, J. et al. Nat. Commun. 12, 2287 (2021), doi: 10.1038/s41467-021-22519-z; Grunewald, J. et al. Nat. Biotechnol. 37, 1041-1048 (2019), doi:10.1038/s41587-019-0236- 6; Rees et al Sci Adv (2019), doi: 10.1126/sciadv.aax5717.
  • amino acid deletion refers to the removal of an amino acid at a particular position in a parent, reference or wild-type (WT) polypeptide sequence.
  • WT wild-type polypeptide sequence
  • D108- or D108A or D108del designates a deletion of aspartic acid at position 108.
  • TadA substitution variant refers to a TadA variant wherein one or more amino acid residue(s) are substituted in the obtained amino acid sequence by another amino acid residue, as compared to the wild-type amino acid sequence (WT TadA).
  • the sequence-targeting protein (ii) and the RNA-ligand binding complex (iii) are capable of recruiting the effector protein (i) to a target site (e.g., a protospacer) in the nucleic acid molecule.
  • Base editing refers to a process wherein a nucleotide base is modified as compared to the initial (e.g., wild-type) base at the same position.
  • Base editing e.g., targeted point mutations
  • Adenosine deaminases remove an amino group from the deoxyadenosine nucleotide target, converting the dA into inosine.
  • inosine is recognized as guanine by polymerase enzymes, resulting in transition (also referred to as "conversion") of an A:T base pair into a G:C base pair in the DNA that has been edited.
  • a target site may comprise 17-20 nucleotides (protospacer) including at least one dA (e.g., located in a position between 4 to 11 within the protospacer).
  • Sequence-targeting protein refers to a protein capable of targeting a specific nucleic acid sequence (e.g., binding to a specific nucleic acid sequence is preferred as compared to nucleic acid sequences having at least one nucleotide difference to the specific nucleic acid sequence).
  • the sequence-targeting protein may be selected from the group consisting of an amino acid sequence motif (e.g., a pentatricopeptide repeat, PPR), a meganuclease (MN), a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN) and a CRISPR-Cas system.
  • PPR pentatricopeptide repeat
  • MN meganuclease
  • ZFN zinc-finger nuclease
  • TALEN transcription activator-like effector nuclease
  • gRNA refers to a nucleic acid comprising a nucleotide sequence (guide sequence) that is complementary to a sequence (target site) of a target nucleic acid, and the nucleic acid of the gRNA forms a complex with a CRISPR-Cas protein.
  • a gRNA comprises, consists essentially of, or consists of a CRISPR RNA (crRNA) and in some embodiments, it may also comprise a trans-activating CRISPR RNA (tracrRNA).
  • sgRNA single guide RNA
  • tracrRNA a contiguous chain of nucleotides
  • each gRNA (or component thereof, e.g., crRNA and tracrRNA if present) may independently be encoded by a vector such as a plasmid, a lentivirus, an adeno associated virus (AAV), a retrovirus, an adenovirus, a coronavirus, a Sendai virus, and the like.
  • a vector such as a plasmid, a lentivirus, an adeno associated virus (AAV), a retrovirus, an adenovirus, a coronavirus, a Sendai virus, and the like.
  • Ligand binding moiety refers to a moiety such as an aptamer e.g., oligonucleotide or peptide or another compound that binds to a specific ligand and can reversibly or irreversibly be associated with that ligand.
  • an aptamer e.g., oligonucleotide or peptide or another compound that binds to a specific ligand and can reversibly or irreversibly be associated with that ligand.
  • To be reversibly associated means that two molecules or complexes can retain association with each other by, for example, non-covalent forces such as hydrogen bonding, and be separated from each other without either molecule or complex losing the ability to associate with other molecules or complexes.
  • the ligand binding moiety links the nucleic acid deaminase and the sequence-targeting protein through the binding of the ligand binding moiety to its ligand.
  • the least one nucleic acid deaminase and the ligand are directly fused or connected, i.e., no linker is present between the deaminase and the ligand.
  • both molecules are connected through a linker.
  • both molecules are linked via other molecules or domains, such as via a SH3 domain, wherein the two molecules are connected through a SH3 (Src 3 homology) domain in one molecule and a SHL (SH3 interaction ligand) in the other molecule.
  • the effector protein and the sequence-targeting protein are directly fused or connected, i.e., no linker is present between the effector and the sequencetargeting protein.
  • both molecules are connected through a linker.
  • both molecules are linked via other molecules or domains, such as via a SH3 domain, wherein the two molecules are connected through a SH3 (Src 3 homology) domain in one molecule and a SHL (SH3 interaction ligand) in the other molecule.
  • RNA-ligand binding complex refers to a complex comprising two subcomponents: (a) an RNA moiety capable of binding to the sequence-targeting protein and (b) an RNA moiety capable of binding to a target site in a nucleic acid molecule.
  • the complex may comprise at least one ligand binding moiety.
  • the ligand binding moiety is an RNA motif.
  • the RNA moiety capable of binding to the sequence-targeting protein is a guide RNA.
  • the RNA moiety capable of binding to a target site in a nucleic acid molecule is a CRISPR motif, such as a tracrRNA.
  • the nucleic acid deaminase comprises or consists of a Staphylococcus aureus ⁇ N TadA enzyme (SEQ ID NO: 3) or a deaminase comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of sequence identity to SEQ ID NO: 3, wherein one, two or three amino acid residue(s) selected from A102, D103 and D104 are deleted.
  • SEQ ID NO: 3 Staphylococcus aureus ⁇ N TadA enzyme
  • the nucleic acid deaminase comprises or consists of a Streptococcus pyogenes WT TadA enzyme (SEQ ID NO: 4) or a deaminase comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of sequence identity to SEQ ID NO: 4, wherein one, two or three amino acid residue(s) selected from A106, S107 and N108 are deleted.
  • SEQ ID NO: 4 a Streptococcus pyogenes WT TadA enzyme
  • the nucleic acid deaminase comprises or consists of a Caulobacter vibrioides (Caulobacter crescentus) WT TadA enzyme (SEQ ID NO: 7) or a deaminase comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of sequence identity to SEQ ID NO: 7, wherein one, two or three amino acid residue(s) selected from the corresponding residues of ecTadA's A106, R107 and D108 are deleted.
  • a Caulobacter vibrioides Caulobacter crescentus WT TadA enzyme
  • the nucleic acid deaminase comprises or consists of a Shewanella putrefaciens strain 4H WT TadA enzyme (SEQ ID NO: 8) or a deaminase comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of sequence identity to SEQ ID NO: 8, wherein one, two or three amino acid residue(s) selected from the corresponding residues of ecTadA's A106, R107 and D108 are deleted.
  • SEQ ID NO: 8 Shewanella putrefaciens strain 4H WT TadA enzyme
  • TadA deaminases have been described in various species, i.e., considered TadA orthologs.
  • the wild-type TadA is a bacterial TadA deaminase.
  • the bacterial TadA deaminase is selected from the group consisting of Escherichia coli TadA, ecTadA (e.g., UniProtKB P68398; SEQ ID NO: 1), Salmonella enterica TadA (e.g., BioCyc database, STM2568; SEQ ID NO: 2), Staphylococcus aureus TadA (e.g., PDB: 2B3J_A; SEQ ID NO: 3), Streptococcus pyogenes TadA (e.g., UniProtKB Q5XE14; SEQ ID NO: 4), Salmonella typhi TadA (e.g., UniProtKB Q8XGY4; SEQ ID NO:
  • the one or more deleted amino acid residue(s) in the WT TadA protein are located within a region of the wild-type TadA contacting the nucleic acid molecule.
  • said region interacting with the nucleic acid molecule to be modified comprises amino acids 105 to 130 of E.coli WT TadA, or a corresponding region in a TadA ortholog: amino acids 116-141 in Salmonella enterica TadA, amino acids 101-126 in Staphylococcus aureus and 105-130 in Streptococcus pyogenes. See Table 1.
  • compositions comprising the nucleic acid deaminase of the present disclosure and a sequence-targeting protein
  • compositions and methods that include one or more of (1) a nucleic acid deaminase protein (also referred to as "TadA deletion variant” or “TadAA variant”), a nucleic acid encoding the deaminase protein, and/or a modified cell comprising the deaminase protein (and/or a nucleic acid encoding the same) and (2) a sequence-targeting protein.
  • a nucleic acid deaminase protein also referred to as "TadA deletion variant” or “TadAA variant”
  • the sequence-targeting protein may be selected from the group consisting of an amino acid sequence motif (e.g., a pentatricopeptide repeat, PPR), a meganuclease (MN), a zinc- finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), and a CRISPR-Cas system.
  • an amino acid sequence motif e.g., a pentatricopeptide repeat, PPR
  • MN meganuclease
  • ZFN zinc- finger nuclease
  • TALEN transcription activator-like effector nuclease
  • CRISPR-Cas system e.g., TALEN
  • compositions and methods that include one or more of (1) a "nucleic acid deaminase” protein (also referred to as “TadA deletion variant” or “TadAA variant”), a nucleic acid encoding the deaminase protein, and/or a modified cell comprising the protein (and/or a nucleic acid encoding the same); and (2) a CRISPR-Cas protein.
  • compositions and methods that include (1) a "nucleic acid deaminase” protein (also referred to as “TadA deletion variant” or “TadAA variant”), a nucleic acid encoding the protein, and/or a modified cell comprising the protein (and/or a nucleic acid encoding the same); and one or more of (2) a CRISPR-Cas system, wherein a Cas protein bound to RNA is responsible for binding to a targeted sequence.
  • a "nucleic acid deaminase” protein also referred to as "TadA deletion variant” or “TadAA variant”
  • a nucleic acid encoding the protein and/or a modified cell comprising the protein (and/or a nucleic acid encoding the same)
  • a CRISPR-Cas system wherein a Cas protein bound to RNA is responsible for binding to a targeted sequence.
  • sequence-targeting protein is part of a fusion protein wherein the sequence-targeting protein is operably linked to a fusion partner with an activity (an effector protein comprising at least one nucleic acid deaminase according to the present disclosure) and may comprise one or more of a nuclear localization sequence (NLS), a linker, and combinations thereof.
  • an effector protein comprising at least one nucleic acid deaminase according to the present disclosure
  • NLS nuclear localization sequence
  • a CRISPR-Cas protein is part of a fusion protein wherein the CRISPR- Cas protein is operably linked to a fusion partner with an activity (an effector protein comprising at least one nucleic acid deaminase according to the present disclosure) and may comprise one or more of a nuclear localization sequence (NLS), a linker, and combinations thereof.
  • an effector protein comprising at least one nucleic acid deaminase according to the present disclosure
  • NLS nuclear localization sequence
  • this fusion protein including the nucleic acid deaminase of the disclosure is organized in one of the following nonlimiting ways (written N terminus to C terminus; the used in the general architecture indicates the presence of an optional linker or a direct peptide bond):
  • the optional linker may be a peptide linker comprising between 1 and 200 amino acids.
  • the peptide linker comprises the amino acid sequence selected from the group consisting of a (GGGGS)n (SEQ ID NOs: 97 and 98), a (G)n (SEQ ID NO: 99), an (EAAAK)n (SEQ ID NOs: 100 and 101), a (GGS)n (SEQ ID NO: 102), an SGSETPGTSESATPES (SEQ ID NO: 103) motif (see, e.g., Guilinger JP et al. Nat. Biotechnol.
  • the nucleic acid deaminase of the disclosure can also be inlaid into the Cas sequence. This configuration is well known in the art and has been described, for example, in Wang et al. Sig Transduct Target Ther 4, 36 (2019) and Nguyen Tran et al. Nat Commun. 2020; 11: 4871.
  • a base editor of the present disclosure comprises an effector protein comprising at least one nucleic acid deaminase according to the present disclosure (TadA deletion variant) linked to a sequence targeting protein via an RNA-ligand binding complex (aptamer-recruitment-dependent base editing system; or RNA scaffold mediated effector recruitment).
  • Said RNA-ligand binding complex may comprise an RNA moiety capable of binding to the sequence-targeting protein, an RNA moiety capable of binding to a target site in a nucleic acid molecule and a ligand-binding moiety.
  • Composition 1 o TadAA, e.g., ecTadAA; o Cas nickase linked to a NLS (e.g., NLS-SpCas9_D10A-NLS) or nuclease-dead Cas protein linked to a NLS (e.g., NLS-SpCas9_D10A+H840A-NLS); and o RNA-ligand binding complex comprising an RNA moiety capable of binding to the Cas protein and an RNA moiety capable of binding to a target site in a nucleic acid molecule (e.g., guide RNA, gRNA).
  • a nucleic acid molecule e.g., guide RNA, gRNA
  • Composition 2 o TadAA linked to a ligand (e.g., ecTadAA-MCP or ecTadAA-Linker-MCP); o Cas nickase linked to a NLS (e.g., NLS-SpCas9_D10A-NLS) or nuclease-dead Cas protein linked to a NLS (e.g., NLS-SpCas9_D10A+H840A-NLS); and o RNA-ligand binding complex comprising an RNA moiety capable of binding to the Cas protein, an RNA moiety capable of binding to a target site in a nucleic acid molecule and a ligand-binding moiety (e.g., gRNA-MS2).
  • a ligand e.g., ecTadAA-MCP or ecTadAA-Linker-MCP
  • Cas nickase linked to a NLS
  • a base editor comprising a TadA deletion variant may further comprise a protein capable of site-specific deamination selected from a cytosine deaminase (e.g., a naturally occurring cytidine deaminase such as APOBEC, AID or CDA; or variants thereof) or an adenine deaminase (e.g., a TadA variant such as a TadA substitution variant or a TadA deletion variant).
  • a cytosine deaminase e.g., a naturally occurring cytidine deaminase such as APOBEC, AID or CDA; or variants thereof
  • an adenine deaminase e.g., a TadA variant such as a TadA substitution variant or a TadA deletion variant.
  • the nucleotide sequence is adjacent to a "protospacer adjacent motif" (PAM) sequence which is recognized by the CRISPR-Cas protein.
  • a protospacer and its adjacent PAM sequence may collectively be referred to as a target site.
  • the Class 2 CRISPR system of 5.
  • pyogenes uses targeted sites having N12-20NGG, where NGG represents the PAM site from 5.
  • pyogenes, and N12-20 represents the 12-20 nucleotides directly 5' to the PAM site.
  • Additional PAM site sequences from other species of bacteria include NGGNG, NNNNGATT, NNAGAA, NNAGAAW, and NAAAAC.
  • Table 3 Examples of Cas proteins and their preferred PAM sequences.
  • a CRISPR-Cas protein (and/or a nucleic acid encoding the CRISPR-Cas protein) can be a naturally existing CRISPR-Cas protein (e.g., a nuclease) or variants thereof (e.g., a nickase, or a dead nuclease), or an engineered CRISPR-Cas protein derived from a naturally occurring (wild-type) protein.
  • a naturally existing CRISPR-Cas protein e.g., a nuclease
  • variants thereof e.g., a nickase, or a dead nuclease
  • an engineered CRISPR-Cas protein derived from a naturally occurring (wild-type) protein e.g., a naturally occurring (wild-type) protein.
  • the sequence-targeting protein is a nuclease comprising at least one catalytically inactive nuclease domain, such as a CRISPR-Cas protein with at least one catalytically inactive nuclease domain (e.g., a nickase) or a nuclease with no catalytically active nuclease domain (e.g., a nuclease-dead CRISPR-Cas protein).
  • a CRISPR-Cas protein with at least one catalytically inactive nuclease domain (e.g., a nickase) or a nuclease with no catalytically active nuclease domain (e.g., a nuclease-dead CRISPR-Cas protein).
  • Cas proteins include but are not limited to: Casl, CasIB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), SauriCas9, Casio, CaslOd, Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl2f, Casl2h, Casl2i, Casl2j, Casl2l (CasBeta), CasMINI, Mad7, CasX, CasY, Cas 13a, Casl4, C2cl, C2c2, C2c3, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA
  • the Cas protein is a Type II Cas protein, such as Cas9.
  • the Cas protein is a Type V Cas protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl2f, Casl2fl, Casl2h, Casl2i, Casl2j, Casl2l, CasMINI and ErCasl2a (MAD7®).
  • Modified versions of Cas proteins that may be used in the present disclosure, include but are not limited to catalytically inactive versions of the Cas protein, such as dCas9 and dCasl2 or Cas versions that have modified attenuated catalytic activity to provide a nicking function, such as nickase nCas9.
  • a nicking enzyme or nickase is an enzyme that cuts one strand of a double-stranded DNA at a specific recognition nucleotide sequence. These enzymes cut only one strand of the DNA duplex, to produce DNA molecules that are "nicked,” rather than cleaved. Further information about Cas proteins can be found, e.g., in Makarova et al. Nat Rev Microbiol. 2020 Feb;18(2):67-83.
  • Non-limiting examples of amino acid sequences of Cas proteins that may be of use in connection with the present disclosure are:
  • a corresponding guide RNA may comprise any one or more of the following: an RNA moiety capable of binding to the sequence-targeting protein, such as a crRNA, an RNA moiety capable of binding to a target site in a nucleic acid molecule, such as a tracrRNA and/or a scoutRNA.
  • Type II CRISPR-Cas systems utilise a guide RNA comprising a crRNA and a tracrRNA (Type II crRNA:tracrRNA guide).
  • the crRNA comprises a sequence that is complementary to a target site in a nucleic acid.
  • the guide RNA may further comprise a tracrRNA that at minimum can hybridise with the crRNA over a range of at least three nucleotides, and when hybridised over that region can retain association with a Type II Cas protein.
  • the guide RNA can be either a single RNA molecule or a complex of multiple RNA molecules.
  • the sequence targeting component comprises a Type II Cas protein
  • the RNA-ligand binding complex comprises a Type II crRNA:tracrRNA guide RNA.
  • Additional PAM site sequences from other species of bacteria include NGGNG, NNNNGATT, NNAGAA, NNAGAAW, and NAAAAC. See, e.g., US 20140273233, WO 2013176772, Cong et al., (2012), Science 339 (6121): 819-823, Jinek et al., (2012), Science 337 (6096): 816-821, Mali et al, (2013), Science 339 (6121): 823-826, Gasiunas et al., (2012), Proc Natl Acad Sci U S A.
  • the tracrRNA further comprises a distal region that does not hybridise with the crRNA, and it may be upstream of the anti-repeat region.
  • the anti-repeat region is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the crRNA repeat region over at least 7 consecutive nucleotides.
  • the repeat region of the abovedescribed programmable crRNA and the anti-repeat region of the tracrRNA are capable of hybridising to form a hybridisation region, herein referred to as a repeat:anti-repeat duplex.
  • the tracrRNA is from Streptococcus pyogenes.
  • the tracrRNA activity and crRNA activity are part of a single continuous strand of nucleotides, known as single guide RNA (or sgRNA).
  • the crRNA may be immediately upstream of the tracrRNA or it may be upstream of the tracrRNA with an intervening sequence or moiety between the tracrRNA and crRNA. If the tracrRNA and crRNA are part of a contiguous strand of nucleotides (sgRNA), there may be a loop region between the tracrRNA and the crRNA of for example 3 to 6 nucleotides, herein referred to as a tetraloop.
  • sgRNA contiguous strand of nucleotides
  • an active portion of a tracrRNA retains the ability to form a complex with a Cas protein, such as Cas9 or dCas9 or nCas9. See, e.g., ⁇ NO 2014/144592.
  • N denote target-specific spacer; N may be any one of "a”, “c”, “g”, or "t/u”).
  • Type V CRISPR-Cas systems may utilise a guide RNA comprising a crRNA (Type V crRNA-only guide), a crRNA and tracrRNA (Type V crRNA:tracrRNA guide), or a crRNA and scoutRNA (Type V crRNA:scoutRNA guide).
  • the guide RNA can be either a single RNA molecule or a complex of multiple RNA molecules.
  • the sequence targeting component of the present disclosure comprises a Type V Cas protein
  • the RNA-ligand binding complex comprises a crRNA guide RNA, a crRNA:tracrRNA guide RNA or a crRNA:scoutRNA guide RNA.
  • Type V CRISPR-Cas systems wherein the sequence-targeting component is a Cas protein, such as Casl2b from Alicylobacillus acidiphilus (AaCasl2b) or uncultured archaeon 1 (Uni) Casl2fl, also referred as Casl2fl, and its evolved derivatives (CasMINI), require a guide RNA comprising a crRNA and a tracrRNA (Type V crRNA:tracrRNA guide).
  • the crRNA also comprises a repeat region.
  • the repeat region hybridises with the anti-repeat region of the tracrRNA, described below, to form a repeat:antirepeat duplex.
  • the repeat region can comprise from about 20 nucleotides to more than about 35 nucleotides.
  • the repeat region is about 28-33 nucleotides in length, such as 31 nucleotides.
  • the Type V crRNA:tracrRNA guide comprises a transactivating CRISPR RNA (tracrRNA).
  • the tracrRNA sequence may comprise from about 40 nucleotides to more than about 100 nucleotides.
  • the tracrRNA can be about 40, 50, 60, 7080, 90 or more than 100 nucleotides in length.
  • the tracrRNA is about 98-100 nucleotides in length, such as 100 nucleotides.
  • the tracrRNA sequence comprises an anti-repeat region and stem loops. Various tracrRNA sequences are known in the art.
  • an active portion of a tracrRNA retains the ability to form a complex with a Type V Cas protein, such as Casl2b or dCasl2b or nCasl2b or Casl2fl or CasMINI.
  • the tracrRNA is from uncultured archaeon 1 (Uni).
  • the tracrRNA activity and crRNA activity are part of a single continuous strand of nucleotides, known as single guide RNA (or sgRNA).
  • the crRNA may be immediately downstream of the tracrRNA or it may be downstream of the tracrRNA with an intervening sequence or moiety between the tracrRNA and crRNA. If the tracrRNA and crRNA are part of a contiguous strand of nucleotides (sgRNA), there may be a loop region between the tracrRNA and the crRNA of for example 3 to 6 nucleotides, herein referred to as a tetraloop.
  • sgRNA contiguous strand of nucleotides
  • the tracrRNA activity and the crRNA comprising the guide RNA are two separate RNA molecules, which together form the functional guide RNA and part of the RNA-ligand binding complex.
  • the molecule with the tracrRNA activity should be able to interact with (usually by base pairing) the molecule with the crRNA activity, to form a two-part Type V crRNA:tracrRNA guide.
  • Type V CRISPR-Cas systems wherein the sequence-targeting component is a Cas protein such as Casl2dl5, require a guide RNA comprising a crRNA and a scoutRNA (crRNA:scoutRNA guide RNA).
  • a guide RNA comprising a crRNA and a scoutRNA (crRNA:scoutRNA guide RNA).
  • the crRNA also comprises a 5' direct-repeat region.
  • This region comprises a conserved 5' nt sequence that hybridises to a complementary sequence of the scoutRNA, described below.
  • the repeat region can comprise from about 20 nucleotides to more than about 35 nucleotides. In an exemplary embodiment, the repeat region is about 28-33 nucleotides in length, such as 31 nucleotides.
  • the Type V crRNA:scoutRNA guide of this disclosure comprises a short-complementary untranslated RNA (scoutRNA).
  • scoutRNA short-complementary untranslated RNA
  • the scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Casl2 enzymes, and in Casl2d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for hybridisation to the crRNA and subsequent enzymatic activity.
  • biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Casl2c-catalyzed pre-crRNA maturation.
  • the scoutRNA may be 40 to 100 nucleotides long.
  • the scoutRNA sequence comprises a crRNA complementary region, an upstream region that is upstream of the crRNA complementary region, and a downstream region that is downstream of the crRNA complementary region.
  • the crRNA complementary region is 5 nucleotides long.
  • the crRNA complementary region may be located at or near the 5' end of the scoutRNA or at or near the 3' end of the scoutRNA or between consecutive nucleotides within the scoutRNA that are neither at or nor the 5' or 3' end of the scoutRNA.
  • self-complementary regions allow for one or more selfhybridisation regions, loops, and bulges, as well as optionally 5' ssRNA overhangs and or no overhangs. In some embodiments, any bulge or bulges of naturally occurring scoutRNAs are preserved even when the ligand binding moiety is attached.
  • the anti-repeat region is at least 80%, at least 85%, at least 90%, at least 95%, or 100% complementary to the Cas association region over at least 5 consecutive nucleotides.
  • the Cas association region of the above-described programmable crRNA and the anti-repeat region of the scoutRNA are capable of hybridising to form a hybridisation region. If the scoutRNA self-hybridises to form one or more hairpin regions, in some embodiments, its anti-repeat region may form a bulge.
  • the RNA that contains both the scoutRNA and the crRNA is capable of retaining association with a Cas RNA binding domain of a Type V Cas protein.
  • the scoutRNA activity and crRNA activity are part of a single continuous strand of nucleotides.
  • the crRNA may be immediately downstream of the scoutRNA or it may be downstream of the scoutRNA with an intervening sequence or moiety between the scoutRNA and crRNA.
  • the intervening sequence or moiety may be the ligand binding moiety, or a nucleotide or non-nucleotide loop region or ethylene glycol spacers such as 18S, 9S or C3.
  • the crRNA:scoutRNA guide RNA may comprise, consist essentially of or consist of a first region of the scoutRNA, the anti-repeat region of the scout RNA, a second region of the scoutRNA, the loop between the scoutRNA, a Cas association region and the targeting region.
  • the scoutRNA activity and the crRNA comprising the guide RNA are two separate RNA molecules, which together form the functional guide RNA and part of the RNA-ligand binding complex.
  • the molecule with the scoutRNA activity should be able to interact with (usually by base pairing) the molecule (crRNA) having the targeting sequence to form a two-part guide crRNA:scoutRNA.
  • Non-limiting examples of scoutRNAs and crRNAs that may be used in a Type V CRISPR-Cas system connection to the present disclosure appear below.
  • CTTAGTTAAG G ATGTTCCAG GTTCTTTCG GG AGCCTTG G CCTTCTCCCTTAACCTATG CC ACTAAT
  • the sequence targeting component comprises a Type V Cas protein such as Casl2dl5 (Fu, B.X.H., Smith, J.D., Fuchs, R.T. et al. Target-dependent nickase activities of the CRISPR-Cas nucleases Cpfl and Cas9. Nat Microbiol 4, 888-897 (2019). https://doi.org/10.1038/s41564-019-0382-0), it is preferable that the RNA-ligand binding complex comprises a Type V crRNA:scoutRNA guide.
  • the disclosure relates to a cell that comprises the nucleic acid deaminase according to the present disclosure or a base editor comprising said nucleic acid deaminase.
  • the disclosure relates to a cell that is a genetically engineered or a modified cell, that has been engineered or modified by the nucleic acid deaminase according to the disclosure or a base editor comprising said nucleic acid deaminase.
  • the disclosure relates to a cell that is a genetically engineered or modified cell, that has been engineered or modified by one of the methods of the disclosure (a modified cell obtainable by methods disclosed herein).
  • the cell has been isolated from a human or non-human subject.
  • the cell can be selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, in invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, a mammalian cell, a pig cell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell.
  • an archaeal cell a bacterial cell, a eukaryotic cell, a eukaryotic single-cell organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algal cell, an animal cell, in invertebrate cell, a vertebrate cell, a fish cell,
  • the cell can be selected from the group consisting of epithelial cells, endothelial cells and mesenchymal cells. In more specific embodiments, the cell can be selected from the group consisting of bone cells, muscle cells, fat cells, nerve cells, etc. and others cells derived from these cells.
  • the cell can be selected from the group consisting of a stem cell, an immune cell and a lymphocyte.
  • the stem cell include embryonic stem cells, Embryonic stem (ES)-like stem cells, fetal stem cells, adult stem cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), multipotent stem cells, oligopotent stem cells, unipotent stem cells and others derived from these cells.
  • the immune cell include a T cell, a B cell, an NK cell, a macrophage, a mixture thereof, and others described herein.
  • the cell is an in vitro cell that is not the human body, at the various stages of its formation and development.
  • composition comprising an effective amount of the cell and a pharmaceutically acceptable carrier.
  • the nucleic acid deaminase variant and/or base editors are delivered to the cells as a nucleic acid deaminase variant is expressed from an mRNA encoding the nucleic acid deaminase variants.
  • promoters for expression of nucleic acids are known and may be used in the practice of the disclosure. Such promoters are selected from constitutive, regulatable and tissue-specific promoters.
  • common promoters for mammalian expression are, e.g., CMV promoter, human U6 (hU6) promoter, SV40 promoter/enhancer, viral LTRs, promoters of constitutively expressed genes (actin, GAPDH), promoters of genes expressed in a tissue-specific manner, promoters of inducible genes (e.g., steroid hormones).
  • a hU6 promoter may be used for expression of gRNA molecules and a CMV promoter may be used for expression of RNA encoding a protein.
  • the nucleic acid molecules comprise a cell type specific promoter.
  • a "cell type specific" promoter is a promoter that primarily drives expression in certain cell types in one or more organs.
  • the nucleic acid sequence contains one or more of a coding region, an open reading frame (ORF), an expression cassette, a promoter/enhancer or terminator region, an untranslated region (UTR), and a cleavage site.
  • ORF open reading frame
  • UTR untranslated region
  • nucleic acid deaminase, base editors, cells and methods disclosed herein have a wide variety of utilities including modifying and editing (e.g., inactivating or activating) a target polynucleotide in a multitude of cell types, such as the cell types listed above.
  • the nucleic acid deaminase, base editors, cells and methods have a broad spectrum of applications in, e.g., research and therapy and it can be used in research and therapy in a wide spectrum of organisms: animals, plants, prokaryotic organisms, fungi, etc.
  • a deactivating mutation may, in some embodiments, generate a premature stop codon in a coding sequence, which results in the expression of a truncated gene product, e.g., a truncated protein lacking the function of the full-length protein.
  • a truncated gene product e.g., a truncated protein lacking the function of the full-length protein.
  • Other diseases that can be treated by correcting a point mutation or introducing a deactivating mutation into a disease-associated gene will be known to those of skill in the art, and the disclosure is not limited in this respect.
  • the target site in the nucleic acid comprises a point mutation associated with a disease or a disorder.
  • the activity of the nucleic acid deaminase results in a correction of the point mutation.
  • the target site in the nucleic acid comprises a G to A point mutation associated with a disease or disorder, and the deamination of the deoxyadenosine results in a sequence that is not associated with a disease or disorder.
  • the disease or disorder to be treated is selected from the group consisting of phenylketonuria, von Willebrand disease, a neoplastic disease, a neoplastic disease associated with a mutant PTEN or BRCA1, alpha- 1 antitrypsin deficiency and Li-Fraumeni syndrome.
  • the nucleic acid deaminase, base editors, cells and methods of the disclosure can be used in cosmetic applications to modify and/or edit (e.g., inactivating or activating) a target polynucleotide, to correct a mutation or introduce desired edits into a target polynucleotide.
  • the nucleic acid deaminase, base editors or cells of the disclosure can be introduced into or, otherwise, applied to the subject for cleansing, beautifying, promoting attractiveness, or altering the appearance.
  • nucleic acid deaminase Other applications of the nucleic acid deaminase, base editors, cells and methods of the disclosure are in biological computing.
  • the nucleic acid deaminase, base editors, cells and methods disclosed herein can be used to generate a transgenic non-human animal or plant having one or more genetic modification(s) of interest.
  • the transgenic non-human animal is homozygous for the genetic modification.
  • the transgenic non- human animal is heterozygous for the genetic modification.
  • the transgenic non-human animal is a vertebrate, for example, a fish (e.g., zebra fish, gold fish, puffer fish, cave fish, etc.), an amphibian (frog, salamander, etc.), a bird (e.g., chicken, turkey, etc.), a reptile (e.g., snake, lizard, etc.), a mammal (e.g., an ungulate, e.g., a pig, a cow, a goat, a sheep, etc.; a lagomorph (e.g., a rabbit); a rodent (e.g., a rat, a mouse); or a non-human primate.
  • a fish e.g., zebra fish, gold fish, puffer fish, cave fish, etc.
  • an amphibian frog, salamander, etc.
  • a bird e.g., chicken, turkey, etc.
  • a reptile e.g., snake,
  • the wild-type (WT) TadA protein predominantly acts on tRNA.
  • the data provided in the below Examples describe the engineering of WT TadA deaminases capable of dA base editing (TadA deletion variant), e.g., for use as base editors.
  • Example 1 Base editing with ecTadA A deletion variants
  • Escherichia coliTad (ecTadA) variants were engineered and used in a base editing system as an adenosine deaminase acting on DNA, e.g., single stranded DNA (ssDNA).
  • ssDNA single stranded DNA
  • ecTadA-D108A One, two or three specific amino acids in
  • the ecTadAA deletion mutations correspond to a specific in-frame deletion of three, six or nine consecutive nucleotides at the DNA level of the WT ecTadA.
  • the nucleic acids encoding the ecTadAA deletion variants were cloned into expression plasmids and expressed in vitro in HEK293T cells, along with a SpCas9 nickase and a site-specific guide RNA.
  • a non-transfected (NT) control was included in this study as a negative control.
  • N-to-G The base editing efficiency data (N-to-G) were expressed as the mean.
  • G-to-G events were excluded from the analysis, shown as (not applicable) in the Figures.
  • the nucleotide sequence corresponding to the different ecTadAA deletion variants was designed in-silico and synthesized as a G-blockTM, which was cloned into an expression plasmid.
  • Plasmids expressing a WT ecTadA or an ecTadAA deletion variant under a CMV promoter were generated. A total of seven constructs were generated, which included an engineered ecTadAA deletion variant with one, two or three amino acid deletions. Each ecTadAA deletion variant contained an NLS sequence N-terminally fused. The regions from amino acids 105-130 of the resulting protein sequences of the seven variants are displayed in Fig. 1 and the corresponding deletion variant named DI through D7.
  • gRNA CRISPR-Cas protein and guide RNA
  • SpCas9 nuclease containing the D10A mutation i.e., an SpCas9 nickase, also referred to as SpCas9_D10A
  • a plasmid was used to express SpCas9 nickase.
  • An SpCas9 gRNA was used to target the SpCas9_D10A to Site2A (SEQ ID NO: 24). The gRNA was expressed under the control of a U6 promoter in a plasmid format.
  • Table 4 Nucleotide sequences of the gRNA targeting Site2a.
  • MS2 hairpin sequence is displayed in italics and bold whilst the targeting region of the gRNA (20 nucleotides) is underlined, "t” represents uracil in the RNA sequence.
  • HEK293T cells were co-transfected in HEK293T cells to provide all three components of the base editing platform: (a) the SpCas9_D10A nickase, (b) the guide RNA targeting the Site2A genomic locus, hereinafter referred to as Site2A, and (c) the respective ecTadAA deletion variant catalysing the A-to-G transition.
  • Site2A the guide RNA targeting the Site2A genomic locus
  • Site2A the respective ecTadAA deletion variant catalysing the A-to-G transition.
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • the medium was removed, and the cells were washed lx with PBS and 50 pl of Trypsin enzyme (Thermo Fisher Scientific) was added to each well. After the cells were dissociated, 20 pl of the resuspended cell solution were transferred to a 96 well plate and were incubated with 60 pl of DirectPCR lysis reagent (Viagen Biotech) under the following conditions: 55°C for 45 minutes followed by 95°C for 15 minutes. The cell lysates were stored at -20 °C until further use.
  • DirectPCR lysis reagent Viagen Biotech
  • Primers used in the PCR are Site2A-Forward (Site2A-F; SEQ ID NO: 87) and Site2A-Reverse (Site2A-R; SEQ ID NO: 88).
  • N-to-G The base editing efficiency data (N-to-G) were expressed as the mean. In the context of N- to-G editing (TadA variants), G-to-G events were excluded from the analysis, shown as (not applicable).
  • Example 2 Base editing with ecTadAA deletion variants fused to an aptamer binding protein os port of on aptamer-recruitment-dependent base editing system
  • the ecTadAA deletion variants described in Example 1 were engineered, fused to an MS2 Coat Protein (MCP) binding protein and used in a base editing system as an adenosine deaminase acting on ssDNA.
  • Said base editing system comprises the recruitment of the ecTadAA deletion variant via an MS2 aptamer on the gRNA that binds to the MCP protein.
  • the ecTadAA deletion variants summarised in Fig. 1 were fused to MCP via a linker (see Table 2) according to the general structure (NLS-TADA-£//V/C'£/?-MCP; see Table 6).
  • the different ecTadAA-MCP deletion variants (Dl-MCP, D2-MCP, D3-MCP, D4-MCP, D5-MCP, D6-MCP or D7-MCP) and WT-MCP were cloned into expression plasmids and expressed in vitro in HEK293T cells, along with a plasmid expressing an SpCas9_D10A and one of four specific gRNA plasmids for each of four targets Site2A, Site2c, Site45 and Site312.
  • These gRNAs contained a 3' RNA aptamer hairpin (MS2 hairpin sequence) which mediates the recruitment of the respective ecTadAA-MCP.
  • a WT-MCP was included in this study as a negative control.
  • Plasmids expressing a wild-type or a mutant ecTadA fused to MCP under a pCMV promoter were generated. A total of seven constructs were generated, which include an engineered ecTadAA deletion variant with one, two or three amino acid deletions. Each ecTadAA deletion variant consists of an NLS sequence fused to the ecTadA N-terminus which is fused to MCP via a linker. The amino acid sequences of the seven ecTadAA-MCP deletion variants are displayed in Table 6.
  • Table 6 Amino acid sequences of the seven TadA deletion variants generated (D1-D7), and the amino acid sequence of the wild-type TadA (WT ecTadA; referred to as "WT") used in the experiments.
  • the general structure is "MA-[NLS]-[ecTadA]-[L]-[MCP]".
  • the N-terminal NLS sequence, [NLS] is highlighted in bold, the linker, [L] is depicted in italic/bold/underlined and the MCP is underlined (NLS-TADA-/./nfcerl- MCP).
  • ecTadA deletion variants ecTadA-D108A (referred to as "DI”); ecTadA-R107A (referred to as “D2”); ecTadA-A106A (referred to as “D3”); ecTadA-D108A-R107A (referred to as “D4"); ecTadA-D108A-R107A-A106A (referred to as "D5"); ecTadA-R107A-A106A (referred to as "D6”); ecTadA-D108A-A106A (referred to as "D7”).
  • SpCas9 nuclease containing the D10A mutation i.e., an SpCas9 nickase, also referred to as SpCas9_D10A
  • a plasmid was used to express SpCas9 nickase.
  • An SpCas9 gRNA scaffold containing an RNA aptamer hairpin (MS2 hairpin sequence) in 3' position was used to target the SpCas9_D10A to the four different genomic targets Site2a (SEQ ID NO: 24), Site2c (SEQ ID NO: 25), Site45 (SEQ ID NO: 26) and Site312 (SEQ ID NO: 27).
  • the gRNAs were expressed under the control of a U6 promoter in a plasmid format. The sequences of the different gRNAs are displayed in Table 7.
  • Table 7 Nucleotide sequences of different gRNAs targeting Site2c, Site2a, Site45 and Site312.
  • MS2 hairpins are displayed in italics and bold whilst the targeting region of the gRNA (20 nucleotides) is underlined, "t” represents uracil in the RNA sequence.
  • HEK293T cells were co-transfected in HEK293T cells to provide all three components of the base editing platform: (a) the SpCas9_D10A nickase, (b) the guide RNA containing the MS2 aptamer and targeting the different sites, and (c) the respective ecTadAA deletion variant fused to MCP and catalysing the A-to-G transition.
  • HEK293T cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U ml-1 penicillin/streptomycin.
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • PCR-amplified targeted regions were Sanger sequenced.
  • N-to-G base editing efficiency
  • N-to-G The base editing efficiency data (N-to-G) were expressed as the mean. In the context of N- to-G editing (TadA variants), G-to-G events were excluded from the analysis, shown as (not applicable).
  • the three variants with only one amino acid deletion were the most reproducibly active variants across the four sites, with notably up to 56% base editing on A5 of Site2c with D3-MCP (see Fig. 3B), up to 48% base editing on A6 of Site45 with D4-MCP (see Fig. 3C) and up to 15% base editing on A7 of Site312 with D4-MCP (see Fig. 3D).
  • Example 3 Base editing with ecTadAA deletion variants or ecTadA substitution variants fused to an aptamer binding protein as part of an aptamer-recruitment-dependent base editing system
  • the ecTadAA-MCP deletion variants (Dl-MCP, D2-MCP, D3-MCP) were tested alongside ecTadA substitution variants which contain a substitution instead of the deletion at the same amino acid position.
  • Dl-MCP ecTadA-D108A
  • D2-MCP ecTadA-R107A
  • ecTadA-R107C ecTadA-R107C-MCP
  • D3-MCP (ecTadA- A106A) was compared to ecTadA-A106V (substitution variant for D3; SEQ ID NO: 52).
  • ecTadAA deletion variants fused to MCP via a linker or ecTadA substitution variants fused to MCP via a linker were cloned into expression plasmids and expressed in vitro in HEK293T cells along with an SpCas9_D10A plasmid and one of four specific gRNA plasmids for each of four targets: Site2a, Site2c, Site45 and Site312.
  • These gRNAs contained a 3' RNA aptamer hairpin (MS2 hairpin sequence) which mediates the recruitment of the respective ecTadAA-MCP deletion variants or ecTadA substitution variants.
  • a non-transfected (NT) control was included in this study as a negative control.
  • MA-NLS-ecTadA-D108N-linker-MCP amino acid sequence ("reference construct for DI”; annotation below: NLS in bold; linker in italics, bold and underlined; MCP in underlined)
  • the three plasmids described above were co-transfected in HEK293T cells to provide all three components of the base editing platform: (a) the SpCas9_D10A nickase, (b) the guide RNA containing the MS2 aptamer and targeting the different sites, and (c) the respective ecTadAA deletion variant or ecTadA substitution variant fused to MCP and catalysing the A-to-G transition.
  • HEK293T cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U ml-1 penicillin/streptomycin. 24 hours prior to transfection 10,000 cells were seeded into a single well of a 96-well plate. After 24 hours the cells were lipid transfected with 200 ng of plasmid DNA (75 ng SpCas9D10A vector, 75 ng ecTadAA-MCP or corresponding ecTadA substitution variant fused to MCP vector, and 50 ng gRNA expression vector) using DharmaFECTTM DUO (DharmaconTM reagents; T-2012-02).
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • PCR-amplified targeted regions were Sanger sequenced.
  • N-to-G base editing efficiency
  • N-to-G The base editing efficiency data (N-to-G) were expressed as the mean. In the context of N- to-G editing (TadA variants), G-to-G events were excluded from the analysis, shown as (not applicable).
  • Example 4 Base editing with homo- and heterodimer ecTadAA deletion variants
  • An ecTadA variant was engineered, fused to either (i) an identical ecTadA variant to generate an ecTadA homodimer or to (ii) WT to generate a heterodimer.
  • the ecTadA variant was then further fused to an MCP binding protein and used in a base editing system as an adenosine deaminase effector.
  • the homo- and heterodimers fused to MCP were recruited via an MS2 aptamer present on the gRNA.
  • Different configurations of the homo- and heterodimer ecTadAA deletion variants fused to MCP were prepared.
  • Fig. 5A and Fig. 5B The schematics of the homo- and heterodimer ecTadAA deletion variants are depicted in the left part of Fig. 5A and Fig. 5B. Base editing efficiency was tested and compared to an ecTadAA deletion variant monomer fused to MCP.
  • the Dl-MCP monomer, N-terminally fused MCP-D1 monomer, D1-D1-MCP homodimer, Dl-WT-MCP heterodimer, N-terminally fused MCP-D1-WT heterodimer or WT-D1-MCP heterodimer were cloned into expression plasmids and expressed in vitro in HEK293T cells along with an SpCas9-D10A plasmid and one of two specific gRNA plasmids for each of two targets Site2c and Site45.
  • These gRNAs contained an RNA aptamer hairpin (MS2 hairpin sequence) in 3' position, which mediates the recruitment of the respective monomer or homo- or heterodimer ecTadAA-MCP deletion variant.
  • N-to-G The base editing efficiency data (N-to-G) were expressed as the mean. In the context of N- to-G editing (TadA variants), G-to-G events were excluded from the analysis, shown as (not applicable).
  • Plasmids expressing the ecTadAA deletion variant (DI) as a monomer or as homo- or heterodimer were generated, under a pCMV promoter. A total of seven constructs were designed, as shown below. Each variant also contained an NLS sequence via a linker (not shown). 1. Dl-MCP monomer with C-terminally fused MCP (SEQ ID NO: 43),
  • WT-D1-MCP heterodimer with C-terminally fused MCP (SEQ ID NO: 58).
  • SpCas9 nuclease containing the D10A mutation i.e., an SpCas9 nickase, also referred to as SpCas9_D10A
  • a plasmid was used to express SpCas9 nickase.
  • An SpCas9 gRNA scaffold containing an RNA aptamer hairpin (MS2 hairpin sequence) in 3' position was used to target the SpCas9_D10A to 2 genomic targets: Site2c (SEQ ID NO: 25) and Site45 (SEQ ID NO: 26).
  • the gRNAs were expressed under the control of a U6 promoter in a plasmid format. The sequences of the different gRNAs are displayed in Table 7 above.
  • HEK293T cells were co-transfected in HEK293T cells to provide all three components of the base editing platform: (a) the SpCas9_D10A nickase, (b) the guide RNA containing the MS2 aptamer and targeting the different sites, and (c) the respective ecTadAA deletion variant or ecTadA substitution variant fused to MCP and catalysing the A-to-G transition.
  • HEK293T cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U ml-1 penicillin/streptomycin.
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • N-to-G The base editing efficiency data (N-to-G) were expressed as the mean. In the context of N- to-G editing (TadA variants), G-to-G events were excluded from the analysis, shown as (not applicable).
  • the NT control shows no editing at Site2c.
  • All the homo- and heterodimer configurations of DI ecTadAA deletion variant show base editing ranging from 6% to 52% A-to-G transition (see Fig. 5A).
  • the highest mean level of editing is with Dl-MCP monomer at position 5 of Site2c.
  • Fig. 5B shows the editing activities of the homo- and heterodimers at Site45. At this site, all variants show editing at position 6, demonstrated by A-to-G conversion. The highest mean level of editing was seen with Dl-MCP.
  • Example 5 Base editing with ecTadAA deletion variants using base editing systems comprising g catalytically inactive SpCgs9 nuclegse.
  • the ecTadAA deletion variant DI C-terminally fused to MCP (Dl-MCP) described in Example 2 was used in combination with a base editing system comprising either a SpCas9_D10A nickase or a nuclease-dead SpCas9.
  • the nuclease-dead SpCas9 is a catalytically inactive SpCas9 enzyme containing a D10A and H840A double-mutation (also referred to as SpCas9_D10A+H840A).
  • the base editing system further comprised a guide RNA specifically targeting either the Site45, SiteB2M or Site2c.
  • the guide RNA contained an RNA aptamer hairpin (MS2 hairpin sequence) in 3' position for the recruitment of Dl-MCP.
  • the individual base editing system components were cloned into expression plasmids and expressed in vitro in HEK293T cells following co-transfection of three individual plasmids.
  • the first plasmid expressed either the SpCas9_D10A nickase or the dead SpCas9
  • the second plasmid expressed the specific guide RNAtargeting eitherSite45, SiteB2M or Site2c that contained MS2, and the third plasmid expressed the ecTadAA deletion variant (Dl- MCP).
  • N-to-G The base editing efficiency data (N-to-G) were expressed as the mean. In the context of N- to-G editing (TadA variants), G-to-G events were excluded from the analysis, shown as (not applicable).
  • ecTadAA deletion variants described in Example 1 were fused to the N-terminus of a Cas9 nickase (nCas9; SpCas9_D10A) to generate ecTadAA-nCas9 fusion base editors.
  • Dl-MCP showed 8% and 30% base editing at the adenosine in position 5 (A5, see Fig. 7A, left column), while ratAPOBECl showed 30% and 53% base editing at the cytidine in position 6 (C6, see Fig. 7A, right column).
  • C6 see Fig. 7A, right column
  • the presence of both Dl-MCP and ratAPOBECl-MCP resulted in a simultaneous base editing at A5 by Dl- MCP and at C6 by ratAPOBECl (see Fig. 7A, last row).
  • Dl-MCP showed 4% and 8% base editing at A5 (see Fig.
  • the ecTadAA deletion variant DI C-terminally fused to MCP (Dl-MCP) described in Example 2 was expressed in vitro in hiPSCs from mRNA along with mRNA expressing an SpCas9_D10A and a synthetic gRNA for the Site2c target.
  • the synthetic gRNA contained a 3' RNA aptamer hairpin (MS2 hairpin sequence) which mediates the recruitment of Dl- MCP.
  • a non-transfected (NT) control was included in this study as a negative control.
  • the ability to perform base editing using a system that utilizes the Dl-MCP variant compared to the NT control was measured via Sanger sequencing 72 hours after electroporation.
  • the measurement of the base editing efficiency from the sequencing data was performed as described in Example 2.
  • Plasmids expressing a Dl-MCP under a T7 promoter were generated.
  • the amino acid sequence of Dl-MCP is displayed in Table 6.
  • mRNA was produced using an in vitro transcription (IVT) assay.
  • DNA plasmids were linearized with Sspl-HF restriction enzyme (New England Biolabs) for 4 hours at 37°C followed by column cleanup with the DNA Clean & Concentrator kit (Zymo Research).
  • the resulting template was used for in vitro transcription with the T7 RiboMAXTM Large Scale RNA Production System (Promega) followed by RQ1 DNase (Promega) treatment for 15 minutes at 37°C.
  • the resulting mRNA was purified with the Monarch® Spin RNA Cleanup Kit (New England Biolabs), aliquoted, and analyzed for quality via the TapeStation High Sensitivity RNA ScreenTape (Agilent).
  • SpCas9 nuclease containing the D10A mutation i.e., an SpCas9 nickase, also referred to as SpCas9_D10A
  • SpCas9_D10A an SpCas9 nuclease containing the D10A mutation
  • An SpCas9 gRNA scaffold containing an RNA aptamer hairpin (MS2 hairpin sequence) in 3' position was used to target the SpCas9_D10A to the genomic target Site2c.
  • the gRNA was synthesized inhouse, and the sequence of the gRNA is displayed in Table 7.
  • Cell culture and electroporation hiPSCs were cultured on Geltrex matrix (Thermo Fisher Scientific)-coated cell culture plates in mTeSRTM PLUS medium (STEMCELL Technologies) at 37°C and 5% CO2. Sub-confluent hiPSC cultures were passaged by non-enzymatic dissociation with GibcoTM VerseneTM solution (Thermo Fisher Scientific) and re-plated as clumps.
  • hiPSC cultures were treated with 10 pM Rho kinase inhibitor (Y-27632, STEMCELL Technologies).
  • hiPSC colonies were dissociated to single cells using GibcoTM StemProTM AccutaseTM reagent (Thermo Fisher Scientific) prior to resuspension in P3 electroporation buffer (Lonza).
  • Dissociated hiPSC samples (250,000 cells per sample) were electroporated with 40 pmol of the gRNA targeting Site2c and 1.6pmol (2.5 pg) of the SpCas9 nickase mRNA and 1.6 pmol (0.75 pg) of the Dl-MCP mRNA using the Amaxa 4D-Nucleofector® System with a 20 pl Nucleocuvette® Strip format (Lonza).
  • hiPSCs were transferred directly to Geltrex matrix-coated cell culture vessels containing pre-warmed mTeSR PLUS medium supplemented with 10 pM Rho kinase inhibitor and incubated at 37°C, 5% CO2 for 24 hours. Culture medium was then changed for mTeSR PLUS medium without additional Rho kinase inhibitor and electroporated hiPSCs were expanded with daily complete medium changes.
  • the medium was removed, and the cells were washed once with PBS and resuspended in 50 pl PBS. After the cells were dissociated, 20 pl of the resuspended cell solution were transferred to a 96 well plate and incubated with 60 pl of DirectPCR® lysis reagent (Viagen Biotech) under the following conditions: 55°C for 45 minutes followed by 95°C for 15 minutes. The cell lysates were stored at -20°C until further use.
  • DirectPCR® lysis reagent Viagen Biotech
  • the present disclosure is useful in a wide range of fields, including research, medicine (e.g., therapeutic treatment, drug discovery), agriculture (e.g., agricultural, fishery and livestock production, breeding etc.), and biological material production.
  • medicine e.g., therapeutic treatment, drug discovery
  • agriculture e.g., agricultural, fishery and livestock production, breeding etc.
  • biological material production e.g., biological material production.

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Abstract

La présente invention concerne des systèmes, des procédés et des compositions pour la modification d'acides nucléiques (par exemple, l'édition de bases) par l'utilisation d'une désaminase d'acide nucléique ou d'un éditeur de bases contenant la désaminase d'acide nucléique. La désaminase d'acide nucléique peut désaminer une désoxyadénosine (dA) dans une molécule d'acide nucléique et comporte une séquence d'acides aminés d'une TadA de type sauvage, caractérisée en ce qu'un ou plusieurs résidus d'acides aminés de la TadA de type sauvage sont supprimés et en ce que le ou les résidus d'acides aminés supprimés sont situés dans une région de la TadA de type sauvage en contact avec la molécule d'acide nucléique.
PCT/EP2025/058591 2024-03-28 2025-03-28 Déaminase d'acide nucleique, éditeur de bases et utilisations associées Pending WO2025202473A1 (fr)

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