WO2020244122A1 - Technique d'édition à base unique de type nouveau et son utilisation - Google Patents

Technique d'édition à base unique de type nouveau et son utilisation Download PDF

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WO2020244122A1
WO2020244122A1 PCT/CN2019/111770 CN2019111770W WO2020244122A1 WO 2020244122 A1 WO2020244122 A1 WO 2020244122A1 CN 2019111770 W CN2019111770 W CN 2019111770W WO 2020244122 A1 WO2020244122 A1 WO 2020244122A1
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amino acid
vector
sequence
gene editing
acid sequence
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杨辉
周昌阳
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中国科学院脑科学与智能技术卓越创新中心
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04001—Cytosine deaminase (3.5.4.1)

Definitions

  • the present invention relates to the field of biotechnology, in particular, to a novel single-base editing technology and its application.
  • DNA base editing methods developed in recent years can directly produce precise point mutations in genomic DNA without double-strand breaks (DSB).
  • Two types of basic editors have been reported: cytosine base editors (CBE, C to T and G to A) and adenine base editors (ABE, A to G, T to C).
  • CBE cytosine base editors
  • ABE adenine base editors
  • its application still has a key problem, namely off-target effect.
  • RNA targeting activity mediated by DNA base editing has not been studied before.
  • cytosine base editor BE3 and the adenine base editor ABE7.10 produced tens of thousands of off-target RNA single nucleotide variants (SNV), while cells without base editing only showed a few hundred. SNV.
  • the ABE7.10 developed by David Liu's laboratory of Harvard University can edit the third to eighth bases of the sgRNA target sequence. If there are other bases beside the target base to be edited, it will be edited non-specifically.
  • the purpose of the present invention is to provide a single-base editing technology with high accuracy, significantly reducing RNA off-target effects, and maintaining effective DNA targeting activity.
  • a mutein of cytosine deaminase APOBEC3A said mutein is a non-natural protein, and said mutein is selected from the group of cytosine deaminase APOBEC3A.
  • One or more amino acids are mutated:
  • the 128th position and the 130th position correspond to the 128th position and the 130th position of the sequence shown in SEQ ID NO:1.
  • the cytosine deaminase APOBEC3A is derived from the species: Homo sapiens.
  • the mutein has the activity of catalyzing the hydrolysis and deamination of cytosine to produce uracil.
  • amino acid sequence of the wild-type APOBEC3A enzyme is shown in SEQ ID NO:1.
  • the 128th arginine (R) is mutated to an amino acid residue other than arginine.
  • the 128th arginine mutation is: alanine (A), glycine (G), phenylalanine (F), aspartic acid (D), cysteine ( C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K) , Methionine (M), Serine (S), Proline (P), Threonine (T), Tryptophan (W), Tyrosine (Y), or Valine (V).
  • the arginine at position 128 is mutated to: leucine (L), valine (V), isoleucine (I) or alanine (A).
  • the 128th arginine (R) is mutated to an alanine (A) residue.
  • the tyrosine at position 130 is mutated to an amino acid residue other than tyrosine.
  • the 130th tyrosine mutation is: alanine (A), glycine (G), phenylalanine (F), aspartic acid (D), cysteine ( C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K) , Methionine (M), Serine (S), Proline (P), Threonine (T), Tryptophan (W), Arginine (R), or Valine (V).
  • the 130th tyrosine mutation is: leucine (L), valine (V), isoleucine (I), alanine (A), or phenylpropanine Acid (F).
  • the tyrosine at position 130 is mutated to a phenylalanine (F) residue.
  • the remaining amino acid sequence of the mutant protein is the same or substantially the same as the sequence shown in SEQ ID NO.:1.
  • the said substantially identical is at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5) amino acids are not the same, wherein, The difference includes amino acid substitution, deletion or addition, and the mutant protein still has the activity of catalyzing the hydrolysis and deamination of adenine to form hypoxanthine.
  • mutant protein is cytosine deaminase APOBEC3A with R128A mutation, and the amino acid sequence of the mutant protein is shown in SEQ ID NO: 2.
  • mutant protein is cytosine deaminase APOBEC3A with Y130F mutation, and the amino acid sequence of the mutant protein is shown in SEQ ID NO: 3.
  • the mutant protein is cytosine deaminase APOBEC3A with R128A and Y130F mutations, and the amino acid sequence of the mutant protein is shown in SEQ ID NO: 4.
  • the amino acid sequence of the mutant protein has at least 80% homology with the sequence shown in SEQ ID NO: 2, 3 or 4, preferably at least 85% or 90%, more preferably It is at least 95%, and most preferably at least 98%, and the homology is ⁇ 202/203 or 99.5%.
  • a gene editing enzyme is provided, and the structure of the gene editing enzyme is shown in formula I:
  • Z1 is the amino acid sequence of the cytosine deaminase APOBEC3A mutant protein according to the first aspect of the present invention.
  • Z2 is the amino acid sequence of nuclease Cas9
  • UMI Uracil DNA glycosylase inhibitor
  • L1, L2 and L3 are each independently an optional connecting peptide sequence
  • Z4 is a non-or nuclear localization signal element (NLS);
  • each "-" is independently a peptide bond.
  • amino acid sequence of L1 is shown in SEQ ID NO: 5.
  • amino acid sequence of L1 is the same or substantially the same as the amino acid sequence shown in SEQ ID NO: 5.
  • amino acid sequence of L2 is shown in SEQ ID NO: 6.
  • amino acid sequence of L2 is the same or substantially the same as the amino acid sequence shown in SEQ ID NO: 6.
  • amino acid sequence of L3 is shown in SEQ ID NO:7.
  • amino acid sequence of L3 is the same or substantially the same as the amino acid sequence shown in SEQ ID NO:7.
  • the source of the nuclease Cas9 is selected from the group consisting of Streptococcus pyogenes, Staphylococcus aureus, mutant of Streptococcus pyogenes, or aureus Coccus mutants.
  • the nuclease Cas9 can be replaced with a Cpf1 nuclease, and the source of the Cpf1 nuclease is selected from the following group: Acidaminococcus, Lachnospiraceae , Acid aminococcus mutants, Chaetomillaceae mutants.
  • amino acid sequence of Z2 is shown in SEQ ID NO: 8.
  • amino acid sequence of Z2 is the same or substantially the same as the amino acid sequence shown in SEQ ID NO: 8.
  • amino acid sequence of Z3 is shown in SEQ ID NO: 11.
  • amino acid sequence of Z3 is the same or substantially the same as the amino acid sequence shown in SEQ ID NO: 11.
  • amino acid sequence of Z4 is shown in SEQ ID NO: 9.
  • amino acid sequence of Z4 is the same or substantially the same as the amino acid sequence shown in SEQ ID NO:9.
  • the said substantially the same is at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5, most preferably 1- 3) Amino acids are not identical, wherein the difference includes substitution, deletion or addition of amino acids.
  • the said substantially identical is that the sequence identity between the amino acid sequence and the corresponding amino acid sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.
  • amino acid sequence of the gene editing enzyme is shown in SEQ ID NO: 10.
  • a polynucleotide which encodes the gene editing enzyme as described in the second aspect of the present invention.
  • polynucleotide is selected from the following group:
  • flank of the ORF of the gene editing enzyme described in the second aspect of the present invention additionally contains auxiliary elements selected from the group consisting of signal peptide, secretory peptide, tag sequence (such as 6His), Or a combination.
  • the signal peptide is a nuclear localization sequence.
  • the polynucleotide is selected from the following group: DNA sequence, RNA sequence, or a combination thereof.
  • a vector which contains the polynucleotide according to the third aspect of the present invention.
  • the vectors include expression vectors, shuttle vectors, and integration vectors.
  • a host cell contains the vector as described in the fourth aspect of the present invention, or its genome integrates the polynucleotide as described in the third aspect of the present invention.
  • the host is a prokaryotic cell or a eukaryotic cell.
  • the prokaryotic cell includes: Escherichia coli.
  • the eukaryotic cell is selected from the group consisting of yeast cells, plant cells, mammalian cells, human cells (such as HEK293T cells), or a combination thereof.
  • a method for single-base site-directed editing of genes including the steps:
  • the first vector contains a first nucleotide construct
  • the first nucleic acid construct has a 5'-3' (5' to 3') formula II structure:
  • P1 is the first promoter sequence
  • X1 is a nucleotide sequence encoding the gene editing enzyme of the second aspect of the present invention.
  • L4 is no or connection sequence
  • X2 is a polyA sequence
  • each "-" is independently a bond or a nucleotide linking sequence.
  • the first promoter is selected from the group consisting of CMV promoter, CAG promoter, PGK promoter, EF1 ⁇ promoter, EFS promoter, or a combination thereof.
  • the first promoter sequence is a CMV promoter.
  • the length of the connecting sequence is 30-120 nt, preferably 48-96 nt, and preferably a multiple of 3.
  • first carrier and the second carrier may be the same or different.
  • the first carrier and the second carrier may be the same carrier.
  • the first vector and/or the second vector further contain an expression cassette for expressing a selection marker.
  • the screening marker is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, red fluorescent protein, blue fluorescent protein, or a combination thereof.
  • the method is non-diagnostic and non-therapeutic.
  • the cells are from the following species: humans, non-human mammals, poultry, plants, or microorganisms.
  • the non-human mammal includes rodents (such as mice, rats, rabbits), cows, pigs, sheep, horses, dogs, cats, and non-human primates (such as monkeys).
  • rodents such as mice, rats, rabbits
  • cows such as cows, pigs, sheep, horses, dogs, cats
  • non-human primates such as monkeys
  • the cell is selected from the group consisting of somatic cells, stem cells, germ cells, non-dividing cells or a combination thereof.
  • the cell is selected from the group consisting of kidney cells, epithelial cells, endothelial cells, nerve cells or a combination thereof.
  • the editing window is the 4th to 7th bases of the 20 base sequence targeted by sgRNA, and the 5th base has the highest editing efficiency. Distributed on both sides is significantly reduced, and the editing window of the non-mutated BE3-hA3A editing system is wider than this method.
  • the editing window is from the 3rd amino acid to the 9th amino acid, and the 5th base has the highest editing efficiency. The lateral distribution gradually decreases.
  • kits comprising:
  • the kit further includes:
  • (a2) A second container, and a second vector in the second container, the second vector containing an expression cassette for expressing sgRNA.
  • the first vector and/or the second vector further contain an expression cassette for expressing a selection marker.
  • first container and the second container may be the same container or different containers.
  • the kit also contains instructions, which describe the following instructions: a method for infecting a cell with the first vector and the second vector to perform single-base site-directed editing of genes in the cell .
  • Figure 1 shows the off-target RNA SNV results of each single-base editing system.
  • APOBEC1 is the cytosine deaminase of BE3.
  • TadA-TadA* wild-type TadA enzyme-evolved TadA heterodimer
  • TadA* modified TadA
  • E DNA targeting efficiency of WT, GFP, TadA-TadA*, ABE7.10 and ABE7.10-site 2.
  • Each group n 3 repeats.
  • Figure 2 shows the characterization of off-target RNA SNV.
  • C Distribution of mutation types in each group. The number indicates the percentage of a certain mutation among all mutations.
  • Non-synonymous mutations induced by ABE7.10 are located on oncogenes and tumor suppressors with the highest editing rate. Gene names are shown in blue, amino acid mutations are shown in red, and single nucleotide conversions are shown in green. The GFP group served as a control for all comparisons. All values are expressed as mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, unpaired t test.
  • Figure 3 shows the results of single-cell RNA SNV analysis of cells transfected with the base editor.
  • A SNV image analyzed by single-cell RNA sequencing method.
  • B The expression pattern of ABE, BE3 or GFP in a single cell from single-cell RNA-seq data.
  • F Distribution of mutation types in each cell. The number indicates the percentage of a certain mutation among all mutations.
  • G, H The ratio of SNV shared between any two samples in the same group. The ratio in each cell is calculated by dividing the number of overlapping SNVs between the two samples by the samples in the row.
  • Figure 4 shows the result of rational design of deaminase to eliminate off-target RNA SNV.
  • A Schematic diagram of BE3 and ABE7.10 variants. All deaminase mutations were performed under the background of BE3/ABE7.10. The point mutation is indicated by the red line.
  • G The representative editing site shows that ABE7.10 (F148A) has reduced the width of the editing window. All values are expressed as mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, unpaired t test.
  • Figure 5 shows a schematic diagram of the plasmid.
  • Figure 6 shows a representative distribution of off-target RNA SNV on chromosomes.
  • A APOBEC1, BE3-site 3, BE3-RNF2; B: TadA-TadA*, ABE7.10-site 1 and ABE7.10-site 2
  • Figure 7 shows the distribution of mutation types for each repeat in all groups. The number indicates the percentage of a certain type of mutation among all mutations.
  • A Distribution of mutation types for each repeat in the GFP group.
  • B Distribution of mutation types for each repeat of APOBEC1 and BE3 groups with or without sgRNA.
  • Figure 8 shows that in all BE3 and ABE7.10 transfection groups, genes containing overlapping off-target RNA SNV were significantly higher than random analog genes. P value was calculated by two-sided Student's t'test.
  • Figure 9 shows the similarity between adjacent off-target RNA SNV sequence and target sequence
  • Figure 10 shows the rate of editing non-synonymous mutations induced by BE3 located on oncogenes and tumor suppressor genes. Single nucleotide conversions are shown in green, amino acid mutations are shown in red, and gene names are shown in blue.
  • Figure 11 shows the ratio of non-synonymous mutations induced by editing ABE7.10 located on oncogenes and tumor suppressor genes. Single nucleotide transitions are shown in green, amino acid mutations are shown in red, and gene names are shown in blue.
  • Figure 12 shows that only off-target RNA SNV was detected in RNA, not DNA.
  • the Sanger sequencing chromatogram showed that only U to C mutations were observed in the RNA of the two highest ranked oncogenes, TOPRS and CSDE1.
  • Figure 13 shows the expression level of the transfection vector in a single cell.
  • the expression levels of GFP, APOBEC1 and TadA-TadA* were quantified in all single cells sequenced.
  • the threshold is indicated by the blue dashed line.
  • the log2 (FPKM+1) thresholds of GFP, BE3 and ABE7.10 are 0.3, 1 and 0.3, respectively. Include cells with expression levels above the threshold for further analysis.
  • Figure 14 shows the mutation type distribution of all single cells.
  • Figure 15 shows the distribution of off-target RNA SNV from all single cells on human chromosomes, and its expression level is higher than the threshold.
  • Figure 16 shows the editing rate of BE3-induced non-synonymous mutations on oncogenes and tumor suppressor genes in single cells. Single nucleotide conversions are shown in green, amino acid mutations are shown in red, and gene names are shown in blue.
  • Figure 17 shows the editing rate of non-synonymous mutations induced by ABE7.10 on oncogenes and tumor suppressor genes located in single cells. Single nucleotide conversions are shown in green, amino acid mutations are shown in red, and gene names are shown in blue.
  • Figure 18 shows a representative distribution of off-target RNA SNV on human chromosomes of engineered BE3 and ABE7.10 variants.
  • Figure 20 shows the distribution of mutation types for each sample of the engineered variants of BE3 and ABE7.10.
  • Figure 21 shows the ratio of shared RNA SNV between any two samples in the engineered variants of BE3 and ABE7.10. Calculate the ratio in each cell by dividing the number of overlapping RNA SNV between the two samples by the number of RNA SNV in the row.
  • Figure 23 shows the homology of TadA enzymes in multiple species.
  • the inventors unexpectedly discovered for the first time that the 128th position of the APOBEC3A fragment in the cytosine base editor BE3-hA3A-related cytosine deaminase (APOBEC3A) After the amino acid residue R is mutated to A (ie APOBEC3A R128A ) or the 130th amino acid residue Y is mutated to F (ie APOBEC3A Y130F ), the gene editing window can be maintained while maintaining effective DNA targeting activity Significantly narrowed, that can significantly improve the accuracy of its gene editing; and, experiments have shown that in the gene editing system with this mutation (ie APOBEC3A R128A or APOBEC3A Y130F ), RNA off-target effects are greatly reduced.
  • the present invention has been completed on this basis.
  • base mutation refers to a substitution, insertion and/or deletion of a base at a certain position in a nucleotide sequence.
  • base substitution refers to the mutation of a base at a certain position in a nucleotide sequence to another different base, such as the mutation of C to T.
  • selection marker gene refers to a gene used to screen transgenic cells or transgenic animals in the transgenic process.
  • the selection marker gene that can be used in this application is not particularly limited, and includes various selection marker genes commonly used in the field of transgenics, representative examples Including (but not limited to): luciferin, or luciferase (such as firefly luciferase, Renilla luciferase), green fluorescent protein, yellow fluorescent protein, red fluorescent protein, or a combination thereof.
  • Cas protein refers to a nuclease.
  • a preferred Cas protein is the Cas9 protein.
  • Typical Cas9 proteins include (but are not limited to): Cas9 derived from Staphylococcus aureus.
  • the Cas9 protein can also be replaced by Cpf1 nuclease, and the source of the Cpf1 nuclease is selected from the following group: Acidaminococcus, Lachnospiraceae, acid aminococcus mutants , Mutants of Laospirillaceae.
  • APOBEC3A is a human cytosine deaminase.
  • APOBEC3A enzyme has the activity of cytosine deaminase and can deaminate cytosine (C) into uracil (Uracil, U).
  • APOBEC3A refers to a protein whose amino acid sequence contains the amino acid sequence of APOBEC3A enzyme that has not been mutated as described in the present invention.
  • the wild-type APOBEC3A enzyme has an amino acid sequence as shown in SEQ ID NO:1.
  • the APOBEC3A (R128A) enzyme has an amino acid sequence as shown in SEQ ID NO: 2.
  • the APOBEC3A (Y130F) enzyme has an amino acid sequence as shown in SEQ ID NO: 3.
  • the APOBEC3A (R128A and Y130F) enzyme has an amino acid sequence as shown in SEQ ID NO:4.
  • gene editing enzyme As used herein, the terms “gene editing enzyme”, “gene editing enzyme of the present invention”, and “APOBEC3A R128A of the present invention” are used interchangeably and refer to the gene editing enzyme with the structure of Formula I as described in the second aspect of the present invention:
  • Z1 is the amino acid sequence of the mutein as described in the first aspect of the present invention.
  • Z2 is the amino acid sequence of nuclease Cas9
  • Uracil DNA glycosylase inhibitor (UGI)
  • L1, L2 and L3 are each independently an optional connecting peptide sequence
  • Z4 is a non-or nuclear localization signal element (NLS);
  • each "-" is independently a peptide bond.
  • amino acid sequence of Z2 is shown in SEQ ID NO: 8.
  • amino acid sequence of Z3 is shown in SEQ ID NO: 11.
  • said L1, L2 and L3 each independently have an amino acid sequence selected from the group consisting of GGS, (GGS) 2 , (GGS) 3 , (GGS) 4 , (GGS) 5 , (GGS) 6 , (GGS) 7 , or a combination thereof.
  • the amino acid sequence of L1 is TPGTSESATPES (SEQ ID NO: 5); the amino acid sequence of L2 is SGGS (SEQ ID NO: 6); the amino acid sequence of L3 is SGGS (SEQ ID NO: 6); ID NO: 7).
  • the Z4 is a nuclear localization signal element (NLS), and the amino acid sequence is PKKKRKV (SEQ ID NO: 9).
  • a typical amino acid sequence of the gene editing enzyme of the present invention is shown in SEQ ID NO: 10.
  • the present invention also includes 50% or more of the sequence shown in SEQ ID NO: 10 of the present invention (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98 % Or more, such as 99%) homologous polypeptides or proteins with the same or similar functions.
  • the "same or similar function” mainly refers to "the activity of catalyzing the hydrolysis and deamination of adenine to form hypoxanthine".
  • the amino acid numbering in the gene editing enzyme of the present invention is based on SEQ ID NO.: 10.
  • the amino acid numbering of the editing enzyme may be misaligned with respect to the amino acid numbering of SEQ ID NO.: 10, such as misaligned positions 1-5 to the N-terminus or C-terminus of the amino acid, and conventional sequence alignment techniques in the art are used in the art.
  • misalignment is within a reasonable range, and should not have homology of 80% (such as 90%, 95%, 98%), with the same or similar genes produced due to the misalignment of amino acid numbering
  • a mutant editing enzyme catalytic activity is not within the scope of the gene editing enzyme of the present invention.
  • the gene editing enzyme of the present invention is a synthetic protein or a recombinant protein, that is, it can be a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, and plants) using recombinant technology. Depending on the host used in the recombinant production protocol, the gene editing enzyme of the present invention may be glycosylated or non-glycosylated. The gene editing enzyme of the present invention may also include or not include the initial methionine residue.
  • the present invention also includes fragments, derivatives and analogs of the gene editing enzyme.
  • fragment refers to a protein that substantially maintains the same biological function or activity of the gene editing enzyme.
  • the gene editing enzyme fragment, derivative or analogue of the present invention may be (i) a gene editing enzyme in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are replaced, and such substitution
  • the amino acid residues of may or may not be encoded by the genetic code, or (ii) gene editing enzymes with substitution groups in one or more amino acid residues, or (iii) mature gene editing enzymes and another compound ( For example, a compound that extends the half-life of a gene editing enzyme, such as polyethylene glycol) is fused to form a gene editing enzyme, or (iv) an additional amino acid sequence is fused to the gene editing enzyme sequence to form a gene editing enzyme (such as a leader sequence or secreted Sequence or used to purify the gene editing enzyme sequence or proprotein sequence, or the formation of fusion protein with antigen IgG fragment).
  • these fragments, derivatives and analogs are within the scope well known to those skilled in the art.
  • conservatively substituted are within the scope well known
  • the gene editing enzyme of the present invention can also be modified.
  • Modification (usually without changing the primary structure) forms include: in vivo or in vitro chemically derived forms of gene editing enzymes such as acetylation or carboxylation.
  • Modifications also include glycosylation, such as those gene editing enzymes produced by glycosylation modification during the synthesis and processing of gene editing enzymes or in further processing steps. This modification can be accomplished by exposing the gene editing enzyme to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase).
  • Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). It also includes gene editing enzymes that have been modified to improve their resistance to proteolysis or optimize their solubility.
  • polynucleotide encoding a gene editing enzyme may include a polynucleotide encoding the gene editing enzyme of the present invention, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or gene editing enzymes having the same amino acid sequence as the present invention.
  • These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • an allelic variant is an alternative form of polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the gene editing enzyme it encodes Function.
  • the present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotide of the present invention under stringent conditions (or stringent conditions).
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, and more Fortunately, hybridization occurs when more than 95%.
  • the gene editing enzyme and polynucleotide of the present invention are preferably provided in an isolated form, and more preferably, are purified to homogeneity.
  • the full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This usually involves cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain a very long fragment.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the method of amplifying DNA/RNA using PCR technology is preferably used to obtain the polynucleotide of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (RACE-cDNA end rapid amplification method) can be preferably used.
  • the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein. And can be synthesized by conventional methods.
  • the amplified DNA/RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.
  • the first vector contains a first nucleotide construct
  • the first nucleic acid construct has a 5'-3' (5' to 3') formula II structure:
  • P1 is the first promoter sequence
  • X1 is a nucleotide sequence encoding the gene editing enzyme of the second aspect of the present invention.
  • L4 is no or connection sequence
  • X2 is a polyA sequence
  • each "-" is independently a bond or a nucleotide linking sequence.
  • the first promoter is selected from the group consisting of CMV promoter, CAG promoter, PGK promoter, EF1 ⁇ promoter, EFS promoter, or a combination thereof.
  • the first promoter sequence is a CMV promoter.
  • the length of the connecting sequence is 30-120 nt, preferably, 48-96 nt, and preferably a multiple of 3.
  • the first carrier and the second carrier may be the same or different.
  • the first carrier and the second carrier may be the same carrier.
  • the first vector and/or the second vector further contain an expression cassette for expressing a selection marker.
  • the selection marker is selected from the following group: green fluorescent protein, yellow fluorescent protein, red fluorescent protein, blue fluorescent protein, or a combination thereof.
  • the method is non-diagnostic and non-therapeutic.
  • the cells are from the following species: humans, non-human mammals, poultry, plants, or microorganisms.
  • the non-human mammals include rodents (such as mice, rats, rabbits), cows, pigs, sheep, horses, dogs, cats, and non-human primates (such as monkeys).
  • the cell is selected from the group consisting of somatic cells, stem cells, germ cells, non-dividing cells or a combination thereof.
  • the cells are selected from the group consisting of kidney cells, epithelial cells, endothelial cells, nerve cells or a combination thereof.
  • the editing window when using the method for gene editing, is the 4th to 7th bases of the 20 base sequence targeted by sgRNA, and the 5th base has the highest editing efficiency.
  • the distribution is significantly reduced, and the editing window of the non-mutated ABE7.10 editing system is wider than this method.
  • the editing window is from the 3rd amino acid to the 9th amino acid, and the 5th base has the highest editing efficiency, which is distributed on both sides. Into a gradually decreasing trend.
  • the inventors also developed a novel gene editing enzyme based on the adenine base editor ABE (for example, ABE7.10 F148A ). Specifically, the present inventors respectively mutated the amino acid residue F at position 148 of the TadA fragment and TadA* fragment in the adenine deaminase (TadA-TadA*) associated with the adenine base editor ABE to A( That is, TadA F148A -TadA* F148A ), the results show that it can significantly narrow its gene editing window while maintaining effective DNA targeting activity, which can significantly improve the accuracy of its gene editing; and, experiments have shown that In the gene editing system with this mutation (ie TadA F148A -TadA* F148A ), the off-target effect of RNA is greatly reduced.
  • TadA F148A -TadA* F148A the off-target effect of RNA is greatly reduced.
  • the editing window of the single-base editing system BE3-hA3A is reduced, which greatly improves the accuracy of single-base editing.
  • the editing window is the 4th to 7th bases of the 20 base sequence targeted by sgRNA, and the 5th base has the highest editing efficiency, and the distribution to both sides is significantly reduced.
  • the editing window of the non-mutated BE3-hA3A editing system is wider than this method.
  • the editing window is from the 3rd amino acid to the 9th amino acid, and the 5th base has the highest editing efficiency, and it is distributed to both sides into a gradually decreasing trend. .
  • BE3-hA3A R128A and BE3-hA3A Y130F almost maintained the editing activity of BE3-hA3A, keeping the same activity in the target editing site.
  • the plasmid was constructed using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to standard protocols.
  • the 293T cells were seeded in a 10cm culture dish, and in Dulbecco's modified Eagle medium (DMEM, Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific) and penicillin/streptomycin at 37°C, 5% CO 2 Under cultivation.
  • the cells were transfected with 30 ⁇ g plasmid using Lipofectamine 3000 (Thermo Fisher Scientific). Three days after transfection, the cells were digested with 0.05% trypsin (Thermo Fisher Scientific) and prepared for FACS.
  • GFP-positive cells were sorted and stored in DMEM or Trizol (Ambion) to determine DNA base editing or RNA-seq.
  • DMEM or Trizol Ambion
  • cells were lysed using a one-step mouse genotyping kit (Vazyme), followed by deep sequencing using Hi-TOM or using EditR 1.0.8 quantitative Sanger sequencing.
  • Vazyme mouse genotyping kit
  • Hi-TOM Hi-TOM
  • EditR 1.0.8 quantitative Sanger sequencing For RNA-seq, ⁇ 500,000 cells are collected and RNA is extracted according to standard protocols, and then converted into cDNA, which is used for high-throughput RNA-seq.
  • RNA-seq high-throughput mRNA sequencing
  • FastQC v0.11.3
  • Trimmomatic v0.36
  • Use STAR v2.5.2b to map qualified reads to the reference genome (Ensemble GRCh38) in a 2-pass mode, and its parameters are implemented by the ENCODE project. Then use the Picard tool (v2.3.0) to sort and mark the duplicates of the mapped BAM file.
  • the refined BAM file uses SplitNCigarReads, IndelRealigner, BaseRecalibrator and HaplotypeCaller tools from GATK (v3.5) to perform segmentation reading, crossing splice junctions, partial rearrangement, basic recalibration and variant calling.
  • filter clusters of at least 5 SNVs. These SNVs are within a 35-base window, and variants with a gene quality score> 25 are retained.
  • the mapping quality score is> 20, Fisher Strand Value (FS>30.0), Qual By depth value (QD ⁇ 2.0), and sequencing depth>20.
  • RNA-seq data First trim the original readings of single-cell RNA-seq data and compare them with GRCh38 human transcriptome (STAR v2.5.2b). After deduplication, GATK software (v3.5) was used to identify RNA SNV from individual cells. Those SNVs detected in single cells with DP ⁇ 20.0, FS ⁇ 30.0 and QD ⁇ 2.0 were retained for downstream analysis.
  • Example 1 Off-target RNA SNV detection for various single-base editing systems
  • CBE CBE, BE3 (APOBEC1-nCas9-UGI) or ABE, ABE7.10 (TadA-TadA*-nCas9), and GFP and with or without Single guide RNA (sgRNA) was transfected into cultured 293T cells. After 72 hours of incubation, cells expressing GFP were collected by FACS and then analyzed by RNA-seq. The experimental results of each group were compared with wild-type (WT, untransfected) samples, and RNA SNV was used in each transfection group (Figure 1A).
  • the 9 groups of transfected cells include expressing GFP, APOBEC1, BE3, BE3 with "site 3" sgRNA, BE3 with "RNF2" sgRNA, TadA-TadA*, ABE7.10, and ABE7 with “site 1" sgRNA. 10. ABE7.10 cells with “site 2" sgRNA ( Figure 5).
  • RNA-seq (two or three repetitions per group) was performed on these samples at an average depth of 125x. Call RNA SNV from RNA-seq data in each replicate, and filter out those identified in any WT cells.
  • RNA SNV was found in GFP transfected cells. Surprisingly, there are more RNA and SNV in the expression of APOBEC1, BE3 without sgRNA, and BE3 with site 3 or RNF2 sgRNA (5-40 times that in cells expressing only GFP). Similarly, a large amount of RNA SNV (5-10 times) was also found in cells expressing TadA-TadA*, ABE7.10 without sgRNA, or ABE7.10 with site 1 or site 2 sgRNA.
  • transfection of APOBEC1 or TadA-TadA* induced a higher amount of RNA SNV than other transfection groups, which means that the increase in SNV in CBE or ABE-treated cells may be caused by It is caused by overexpression of APOBEC1 or TadA.
  • off-target RNA SNV was characterized for each single-base editing system.
  • RNA SNV identified in BE3-treated cells is a mutation from G to A or C to U, which is significantly higher than that of GFP-transfected cells ( Figure 2A and 2C and Figure 7) ).
  • This mutation deviation is the same as APOBEC1 itself, indicating that these mutations are not spontaneous, but induced by BE3 or APOBEC1.
  • the GFP group also showed some deviations for A to G and U to C mutations (as shown in Figure 2C), which may be due to innate mutation preference.
  • off-target RNA and SNV induced by CBE and ABE are sgRNA-independent and caused by the overexpression of APOBEC1 and TadA-TadA*, respectively.
  • Example 3 Single-cell RNA SNV analysis of cells transfected with single-base editing system
  • single-cell RNA-seq sequencing was performed on four groups of cells (WT, GFP, BE3-site 3 and ABE7.10-site 1) to avoid random off-target signal loss due to population averaging .
  • BE3 (APOBEC1) transfected cells compared with BE3 (APOBEC1) transfected cells, BE3 (hA3A) transfected 293T cells showed significantly reduced off-target RNA SNV, while maintaining high targeted DNA editing efficiency (Figure 4B and 4C, Figure 4C). 18).
  • the point mutation W90A was introduced into the predicted RNA binding domain of APOBEC1, and it was found that although BE3 (W90A) eliminated the RNA off-target effect, the targeted DNA editing activity of BE3 (W90A) basically did not exist ( Figure 4B and 4C, Figure 18); point mutations Y130F and R128A were introduced into the predicted RNA binding domain of APOBEC3A, and it was found that BE3-hA3A (Y130F) and BE3-hA3A (R128A) can eliminate RNA off-target effects, but BE3-hA3A (Y130F) ) And the targeted DNA editing activity of BE3-hA3A (R128A) remained basically unchanged ( Figure 4B and 4C, Figure 18).
  • the engineered BE3-hA3A R128A and BE3-hA3A Y130F in the present invention have greater application prospects.

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Abstract

La présente invention concerne une enzyme d'édition de gène, caractérisée en ce que la structure de l'enzyme d'édition de gène est telle que représentée par la formule I : Z1-L1-Z2-L2-Z3-Z4 (I), dans laquelle Z1 est la séquence d'acides aminés d'une cytosine désaminase APOBEC3A ; Z2 est la séquence d'acides aminés de spCas9(n) ; et Z1 a une mutation A correspondant au résidu R en position 128 de la séquence telle que représentée dans SEQ ID NO : 1 ; Z3 est la séquence codant pour l'inhibiteur d'Uracile ADN Glycosylase (UGI) ; L1 et L2 sont chacun indépendamment une séquence peptidique de liaison éventuelle ; Z4 n'existe pas ou est un élément de signal de localisation nucléaire (NLS) ; et chaque "-" est indépendamment une liaison peptidique. L'invention concerne un procédé d'édition dirigée sur site d'une base unique dans un gène. Le procédé présente une grande précision dans l'édition d'ADN, et peut réduire de manière significative les effets hors cible de l'ARN.
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