WO2020199200A1 - New-type single-base editing technique and use thereof - Google Patents

Abstract

Provided in the present invention are a new-type single-base editing technique and the use thereof. Specifically provided in the present invention is a gene editing enzyme, characterized in that the structure of the gene editing enzyme is as shown in formula I: Z1-L1-Z2-L2-Z3-L3-Z4 (I), wherein Z1 is the amino acid sequence of the adenine deaminase TadA; Z2 is the amino acid sequence of the TadA* enzyme; and Z1 and/or Z2 have/has a mutation corresponding to the F residue at position 147 and/or 148 of the sequence shown in SEQ ID NO:1; Z3 is the coding sequence of Cas9 nuclease; L1, L2 and L3 are each independently an optional connecting peptide sequence; Z4 is a non- or nuclear localization signal element (NLS); and each "-" is independently a peptide bond. Further provided in the present invention is a method for the site-directed editing of single-base genes. In the method of the present invention, the accuracy of DNA editing is high, and an RNA off-target effect can be significantly reduced.

Description

A new single-base editing technique and its application Technical field

The present invention relates to the field of biotechnology, in particular, to a novel single-base editing technology and its application.

Background technique

Since 2013, the new generation of gene editing technology represented by CRISPR/Cas9 has entered various experiments in the field of biology, which is changing the traditional methods of gene manipulation.

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). However, its application still has a key problem, namely off-target effect.

Previous studies have focused on evaluating off-target mutations in genomic DNA. Recent research results indicate that CBEs rather than ABEs induce a large number of off-target single-nucleotide mutations in the process of gene editing, emphasizing the need to develop higher-fidelity single-base editing tools. In addition to DNA targeting activity, commonly used single-base editing systems may mutate RNA. For example, it was found that the cytosine deaminase APOBEC1 related to CBE can target both DNA and RNA, and it was found that TadA, the adenine deaminase related to ABE, can also induce site-specific inosine formation on RNA. However, the RNA targeting activity mediated by DNA base editing has not been studied before. Studies have shown that both the 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.

At present, among the existing DNA base editing methods, the accuracy of DNA editing is not high, that is, the gene editing window is too large. 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.

Therefore, there is an urgent need in the art to develop a single-base editing technology with high accuracy, significantly reducing RNA off-target effects, and maintaining effective DNA targeting activity.

Summary of the invention

The purpose of the present invention is to provide a single-base editing technique with high accuracy, significantly reducing RNA off-target effects, and maintaining effective DNA targeting activity.

In the first aspect of the present invention, there is provided a mutein of adenine deaminase TadA, said mutein is a non-natural protein, and said mutein is one selected from the group consisting of adenine deaminase TadA Or multiple amino acids are mutated:

Phenylalanine (F) at position 147 and Phenylalanine (F) at position 148;

Wherein, the 147th and 148th positions correspond to the 147th and 148th positions of the sequence shown in SEQ ID NO:1.

In another preferred example, the adenine deaminase TadA is derived from a species selected from the group consisting of Escherichia coli (E. coli), A. aeolicus, and B. subtilis (B. subtilis). ), Yeast CDD1.

In another preferred embodiment, the mutant protein has the activity of catalyzing the hydrolysis and deamination of adenine to form hypoxanthine.

In another preferred example, the adenine deaminase TadA includes TadA* enzyme and wild-type TadA enzyme.

In another preferred embodiment, the adenine deaminase TadA is TadA* enzyme.

In another preferred example, the amino acid sequence of the wild-type TadA enzyme is shown in SEQ ID NO:1.

In another preferred embodiment, the amino acid sequence of the TadA* enzyme is shown in SEQ ID NO: 2.

In another preferred embodiment, the phenylalanine (F) at position 147 is mutated to an amino acid residue other than phenylalanine.

In another preferred embodiment, the mutation of phenylalanine at position 147 is: alanine (A), glycine (G), arginine (R), 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).

In another preferred embodiment, the mutation of phenylalanine at position 147 is: leucine (L), valine (V), isoleucine (I), alanine (A), or tyrosine Acid (Y).

In another preferred example, the phenylalanine (F) at position 148 is mutated to an amino acid residue other than phenylalanine.

In another preferred embodiment, the mutation of phenylalanine at position 148 is: alanine (A), glycine (G), arginine (R), 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).

In another preferred embodiment, the mutation of phenylalanine at position 148 is: leucine (L), valine (V), isoleucine (I), alanine (A), or tyrosine Acid (Y).

In another preferred embodiment, except for the mutations (such as amino acids 147 and 148), 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.

In another preferred embodiment, 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.

In another preferred embodiment, when the adenine deaminase TadA is a wild-type TadA enzyme, the amino acid sequence of the mutant protein is shown in SEQ ID NO: 3.

In another preferred embodiment, when the adenine deaminase TadA is the TadA* enzyme, the amino acid sequence of the mutant protein is shown in SEQ ID NO: 4.

In another preferred embodiment, the amino acid sequence of the mutant protein has at least 80% homology with the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, preferably at least 85% or 90%, and more It is preferably at least 95%, most preferably at least 98%, and the homology is ≤166/167 or 99.4%.

In the second aspect of the present invention, a gene editing enzyme is provided, and the structure of the gene editing enzyme is shown in formula I:

Z1-L1-Z2-L2-Z3-L3-Z4 (I)

among them,

Z1 is the amino acid sequence of adenine deaminase TadA;

Z2 is the amino acid sequence of TadA* enzyme;

And said Z1 and/or Z2 is the amino acid sequence of the mutant protein according to the first aspect of the present invention;

Z3 is the coding sequence of Cas9 nuclease;

L1, L2 and L3 are each independently an optional connecting peptide sequence;

Z4 is a non-or nuclear localization signal element (NLS);

And each "-" is independently a peptide bond.

In another preferred embodiment, the Z1 has the amino acid sequence of wild-type TadA enzyme.

In another preferred embodiment, the Z1 has the amino acid sequence of the wild-type TadA enzyme with F147A and/or F148A mutation.

In another preferred example, the Z1 is a wild-type TadA enzyme with F147A and/or F148A mutations.

In another preferred embodiment, the amino acid sequence of Z1 is shown in SEQ ID NO: 3.

In another preferred embodiment, the Z2 has the amino acid sequence of TadA* enzyme.

In another preferred embodiment, the Z2 has the amino acid sequence of the TadA* enzyme with F147A and/or F148A mutation.

In another preferred embodiment, the Z2 is a TadA* enzyme with F147A and/or F148A mutations.

In another preferred embodiment, the amino acid sequence of Z2 is shown in SEQ ID NO: 4.

In another preferred embodiment, the amino acid sequence of L1 is shown in SEQ ID NO: 5.

In another preferred embodiment, the amino acid sequence of L1 is the same or substantially the same as the amino acid sequence shown in SEQ ID NO: 5.

In another preferred embodiment, the amino acid sequence of L2 is shown in SEQ ID NO: 6.

In another preferred embodiment, the amino acid sequence of L2 is the same or substantially the same as the amino acid sequence shown in SEQ ID NO: 6.

In another preferred embodiment, the amino acid sequence of L3 is shown in SEQ ID NO:7.

In another preferred embodiment, the amino acid sequence of L3 is the same or substantially the same as the amino acid sequence shown in SEQ ID NO:7.

In another preferred embodiment, in the Z3, the source of the Cas9 nuclease is selected from the group consisting of Streptococcus pyogenes, Staphylococcus aureus, mutant of Streptococcus pyogenes, or aureus Coccus mutants.

In another preferred example, in the Z3, the Cas9 nuclease can be replaced with Cpf1 nuclease, and the source of the Cpf1 nuclease is selected from the following group: Acidaminococcus, Lachnospiraceae , Acid aminococcus mutants, Chaetomillaceae mutants.

In another preferred embodiment, the amino acid sequence of Z3 is shown in SEQ ID NO: 8.

In another preferred embodiment, the amino acid sequence of Z3 is the same or substantially the same as the amino acid sequence shown in SEQ ID NO: 8.

In another preferred embodiment, the amino acid sequence of Z4 is shown in SEQ ID NO: 9.

In another preferred embodiment, the amino acid sequence of Z4 is the same or substantially the same as the amino acid sequence shown in SEQ ID NO:9.

In another preferred example, 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.

In another preferred embodiment, 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%.

In another preferred embodiment, the amino acid sequence of the gene editing enzyme is shown in SEQ ID NO: 10.

In the third aspect of the present invention, a polynucleotide is provided, which encodes the gene editing enzyme as described in the second aspect of the present invention.

In another preferred embodiment, the polynucleotide is selected from the following group:

(a) A polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 10;

(b) A polynucleotide whose nucleotide sequence is ≥95% (preferably ≥98%) with the sequence identity of the polynucleotide sequence of (a);

(c) A polynucleotide complementary to the polynucleotide described in any one of (a) and (b).

In another preferred example, the 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.

In another preferred embodiment, the signal peptide is a nuclear localization sequence.

In another preferred embodiment, the polynucleotide is selected from the following group: DNA sequence, RNA sequence, or a combination thereof.

In the fourth aspect of the present invention, a vector is provided, which contains the polynucleotide according to the third aspect of the present invention.

In another preferred embodiment, the vectors include expression vectors, shuttle vectors, and integration vectors.

In the fifth aspect of the present invention, a host cell is provided, the host cell contains the vector according to the fourth aspect of the present invention, or its genome integrates the polynucleotide according to the third aspect of the present invention.

In another preferred embodiment, the host is a prokaryotic cell or a eukaryotic cell.

In another preferred embodiment, the prokaryotic cell includes: Escherichia coli.

In another preferred embodiment, 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.

In the sixth aspect of the present invention, a method for single-base site-directed editing of genes is provided, including the steps:

(i) Provide a cell and a first vector and a second vector, wherein the first vector contains an expression cassette for the gene editing enzyme according to the second aspect of the present invention, and the second vector contains an expression cassette for expressing sgRNA ；

(ii) Infecting the cell with the first vector and the second vector, thereby performing single-base site-directed editing in the cell.

In another preferred example, wherein the first vector contains a first nucleotide construct, and the first nucleic acid construct has a 5'-3' (5' to 3') formula II structure:

P1-X1-L4-X2 (II)

Wherein, 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;

Also, each "-" is independently a bond or a nucleotide linking sequence.

In another preferred example, the first promoter is selected from the group consisting of CMV promoter, CAG promoter, PGK promoter, EF1α promoter, EFS promoter, or a combination thereof.

In another preferred example, the first promoter sequence is a CMV promoter.

In another preferred example, the length of the connecting sequence is 30-120 nt, preferably 48-96 nt, and preferably a multiple of 3.

In another preferred embodiment, the first carrier and the second carrier may be the same or different.

In another preferred embodiment, the first carrier and the second carrier may be the same carrier.

In another preferred embodiment, the first vector and/or the second vector further contain an expression cassette for expressing a selection marker.

In another preferred embodiment, 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.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In another preferred embodiment, the cells are from the following species: humans, non-human mammals, poultry, plants, or microorganisms.

In another preferred embodiment, the non-human mammal includes rodents (such as mice, rats, rabbits), cows, pigs, sheep, horses, dogs, cats, and non-human primates (such as monkeys).

In another preferred embodiment, the cell is selected from the group consisting of somatic cells, stem cells, germ cells, non-dividing cells or a combination thereof.

In another preferred embodiment, the cells are selected from the group consisting of kidney cells, epithelial cells, endothelial cells, nerve cells or a combination thereof.

In another preferred example, when using the method for gene 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. Distributed on both sides is significantly reduced. 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. The lateral distribution gradually decreases.

In the seventh aspect of the present invention, a kit is provided, the kit comprising:

(a1) A first container, and a first vector located in the first container, the first vector containing the expression cassette of the gene editing enzyme according to the second aspect of the present invention.

In another preferred embodiment, 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.

In another preferred embodiment, the first vector and/or the second vector further contain an expression cassette for expressing a selection marker.

In another preferred embodiment, the first container and the second container may be the same container or different containers.

In another preferred embodiment, the kit also contains instructions, which describe the following instructions: a method for infecting cells with the first vector and the second vector to perform single-base-directed editing of genes in the cell .

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

Description of the drawings

Figure 1 shows the off-target RNA SNV results of each single-base editing system.

A: Experimental design plan.

B: WT (n=3 repeats), GFP (n=3), APOBEC1 (n=3 repeats), BE3 (n=3 repeats) and BE3-site 3 (n=2 repeats) DNA Targeting efficiency. Note that APOBEC1 is the cytosine deaminase of BE3.

C: DNA targeting efficiency of WT, GFP, APOBEC1, BE3 and BE3-RNF2. Each group n=3 repeats.

D: DNA targeting efficiency of WT, GFP, TadA-TadA*, ABE7.10 and ABE7.10-site 1. Each group n=3 repeats. Note that TadA-TadA* (wild-type TadA enzyme-evolved TadA heterodimer) is an adenine deaminase of ABE7.10, and modified TadA is represented by TadA*.

E: DNA targeting efficiency of WT, GFP, TadA-TadA*, ABE7.10 and ABE7.10-site 2. Each group n=3 repeats.

F, G: Comparison of off-target RNA SNV between BE3 and ABE7.10 groups.

H: Representative distribution of off-target RNA SNV on human chromosomes of GFP, BE3 and ABE7.10. Chromosomes are represented by different colors. 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 2 shows the characterization of off-target RNA SNV.

A: GFP (n=6 repeats), APOBEC1 (n=3 repeats), BE3 (n=3 repeats), BE3-site 3 (n=2 repeats) and BE3-RNF2 (n=3 times) Repeat) the ratio of G>A and C>U mutations.

B: GFP (n=6 repeats), TadA-TadA* (n=3 repeats), ABE7.10 (n=3 repeats), ABE7.10-site 1 (n=3 repeats) and ABE7 .10-The ratio of A>G and U>C mutations at position 2 (n=3 repetitions).

C: Distribution of mutation types in each group. The number indicates the percentage of a certain mutation among all mutations.

D: The ratio of shared RNA SNV between any two samples in the BE3 and ABE7.10 groups. 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.

E: 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.

C: Detected in single cells treated with GFP-(n=15 cells), BE3-site 3-(n=4 cells) and ABE7.10-site 1-(n=9 cells) The number of off-target RNA SNV.

D: The ratio of G>A and C>U mutations.

E: Ratio of A>G and U>C mutations in GFP (n=15 cells), BE3-site 3 (n=4 cells) and ABE7.10-site 1 (n=9 cells).

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.

I: Editing rate of SNV located on cancer-related genes in at least 3 single cells edited by ABE7.10. 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 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.

B: GFP (n=3 repeats), BE3-site 3 (n=2 repeats), BE3 (hA3A)-site 3 (n=3 repeats) and BE3 (W90A)-site 3 (n = 3 replicates) targeting efficiency.

C: Comparison of off-target RNA SNV in the BE3-site 3 treatment group.

D: Targeting efficiency of GFP, ABE7.10-site 1, ABE7.10 (D53G)-site 1, and ABE7.10 (F148A)-site 1 group. Each group n=3 repeats.

E: Comparison of off-target RNA SNV in ABE7.10 treatment group.

F: Compare the editing efficiency of ABE7.10 and ABE7.10 (F148A) at four different positions. Each group n=3 repeats.

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.

C: Distribution of mutation types for each repetition of TadA-TadA* and ABE7.10 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 conversions 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.

A: Distribution of mutation types in GFP-transfected single cells (n=16 cells).

B: Distribution of mutation types of single cells (n=31 cells) transfected with BE3 site 3. Cells expressing APOBEC1 above the threshold are included in the red square.

C: Distribution of mutation types of ABE7.10-site1-transfected single cells (n=28 cells). Cells with an expression level of TadA-TadA* above the threshold are included in the red squares. This number represents the percentage of a certain mutation among all mutations. SC stands for single cell.

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.

A: The distribution of off-target RNA SNV on human chromosomes of single cells (n=15) transfected with GFP.

B: The distribution of off-target RNA SNV on human chromosomes of single cells (n=4) transfected with BE3 site 3.

C: The distribution of off-target RNA SNV on the human chromosomes of ABE7.10-site 1-transfected single cells (n=9).

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 19 shows the average distribution of mutation types of engineered variants of BE3 and ABE7.10, with n=3 in each group.

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 22 shows a comparison of the width of the edit window between ABE7.10 (n=3) and ABE7.10 ^F148A (n=3).

Figure 23 shows the homology of TadA enzymes in multiple species.

detailed description

After extensive and in-depth research and extensive screening, the inventors unexpectedly discovered for the first time that the TadA fragment and the TadA* fragment in the adenine deaminase (TadA-TadA*) associated with the adenine base editor ABE After mutating the amino acid residue F at position 148 to A (that is, TadA ^F148A -TadA* ^F148A ), the gene editing window can be significantly narrowed while maintaining effective DNA targeting activity, which can significantly increase The accuracy of its gene editing; and, experiments have proved that in the gene editing system with this mutation (ie, TadA ^F148A -TadA* ^F148A ), the off-target effect of RNA is greatly reduced. The present invention has been completed on this basis.

the term

As used herein, the term "base mutation" refers to a substitution, insertion and/or deletion of a base at a certain position in a nucleotide sequence.

As used herein, the term "base substitution" refers to the mutation of a base at a certain position in the nucleotide sequence to another different base, such as the mutation of A to G.

As used herein, "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.

As used herein, the term "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. In the present invention, 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.

Adenine Deaminase TadA

TadA is a prokaryotic RNA editing enzyme.

TadA enzyme has the activity of adenine deaminase and can deaminate adenine (Adenosine, A) into hypoxanthine (Inosine, I). Recombinant TadA protein forms a homodimer, which produces inosine by deaminating adenosine residues at the swing position of tRNA Arg-2.

As shown in Figure 23, TadA has high homology among multiple species. For example, E. coli tadA shows sequence similarity to the yeast tRNA deaminase subunit Tad2p.

In many species, especially at position 148 corresponding to the sequence shown in SEQ ID NO:1 of the present invention, there are highly conserved amino acid residues.

As used herein, the terms "TadA7.10" and "TadA*" are used interchangeably and refer to a mutant based on the amino acid sequence of the wild-type TadA enzyme of the present invention. The mutant amino acid residues include W23R, H36L, P48A, R51L, L84F, A106V, D108N, H123Y, S146C, D147Y, R152P, E155V, I156F and K157N.

Correspondingly, the terms "ABE7.10" and "TadA-TadA*" are used interchangeably, and refer to the amino acid sequence that contains the amino acid sequence of the wild-type TadA enzyme and the TadA* enzyme that have not been mutated according to the present invention. protein.

In one embodiment of the present invention, the wild-type TadA enzyme has the amino acid sequence shown in SEQ ID NO: 1, and the TadA* enzyme has the amino acid sequence shown in SEQ ID NO: 2.

Gene editing enzyme of the present invention and its encoding nucleic acid

As used herein, the terms "gene editing enzyme", "gene editing enzyme of the present invention", "TadA ^F148A- TadA* ^{F148A of the} present invention", and "ABE7.10 ^F148A " are used interchangeably and refer to the second aspect of the present invention. The gene editing enzyme with the structure of formula I:

Z1-L1-Z2-L2-Z3-L3-Z4 (I)

among them,

Z1 is the amino acid sequence of adenine deaminase TadA;

Z2 is the amino acid sequence of TadA* enzyme;

And said Z1 and/or Z2 is the amino acid sequence of the mutant protein according to the first aspect of the present invention;

Z3 is the coding sequence of Cas9 nuclease;

L1, L2 and L3 are each independently an optional connecting peptide sequence;

Z4 is a non-or nuclear localization signal element (NLS);

And each "-" is independently a peptide bond.

In a preferred embodiment, the amino acid sequence of Z1 is an amino acid sequence with F148A mutation at position 148 based on the amino acid sequence shown in SEQ ID NO:1.

In a preferred embodiment, the amino acid sequence of Z2 is based on the amino acid sequence shown in SEQ ID NO: 2, an amino acid sequence in which the F148A mutation occurs at position 148.

In a preferred embodiment, the amino acid sequence of Z3 is shown in SEQ ID NO: 8.

In one embodiment of the present invention, 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.

In a preferred embodiment, the amino acid sequence of L1 is SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 5); the amino acid sequence of L2 is SGGSSGGSSGSETPGTSESATPESSGGSSGGSGS (SEQ ID NO: 6); the amino acid sequence of L3 is SGGS (SEQ ID NO: 6) ID NO: 7).

In a preferred embodiment, the Z4 is a nuclear localization signal element (NLS), and the amino acid sequence is PKKKRKV (SEQ ID NO: 9).

In a preferred embodiment of the present invention, 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".

It should be understood that the amino acid numbering in the gene editing enzyme of the present invention is based on SEQ ID NO.: 10. When the homology between a specific gene editing enzyme and the sequence shown in SEQ ID NO.: 10 reaches 80% or more, the gene 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. The skilled person can generally understand that such 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. As used herein, the terms "fragment", "derivative" and "analog" refer 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). According to the teachings herein, these fragments, derivatives and analogs are within the scope well known to those skilled in the art. In the present invention, conservatively substituted amino acids are preferably generated by amino acid substitutions according to Table I.

Table I

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

Trp(W)	Tyr; phe	Tyr
Tyr(Y)	Trp; phe; Thr; Ser	Phe
Val(V)	Ile; Leu; Met; phe; Ala	Leu

In addition, 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.

The term "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. As known in the art, 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). In the present invention, "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. For the PCR amplification 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.

Once the relevant sequence is obtained, 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.

In addition, 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.

At present, 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. In addition, 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.

Method of the invention

In the present invention, there is also provided a method for single-base site-directed editing of genes, including the steps:

(i) Provide a cell and a first vector and a second vector, wherein the first vector contains an expression cassette for the gene editing enzyme according to the second aspect of the present invention, and the second vector contains an expression cassette for expressing sgRNA ；

(ii) Infecting the cell with the first vector and the second vector, thereby performing single-base site-directed editing in the cell.

In another preferred example, wherein the first vector contains a first nucleotide construct, and the first nucleic acid construct has a 5'-3' (5' to 3') formula II structure:

P1-X1-L4-X2 (II)

among them,

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;

Also, each "-" is independently a bond or a nucleotide linking sequence.

Wherein, the first promoter is selected from the group consisting of CMV promoter, CAG promoter, PGK promoter, EF1α promoter, EFS promoter, or a combination thereof. In a preferred embodiment, the first promoter sequence is a CMV promoter.

In one embodiment of the present invention, the length of the connecting sequence is 30-120 nt, preferably, 48-96 nt, and preferably a multiple of 3.

In the method, the first carrier and the second carrier may be the same or different. In a preferred embodiment, the first carrier and the second carrier may be the same carrier.

Preferably, 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.

In one embodiment of the invention, the method is non-diagnostic and non-therapeutic.

In the method of the present invention, the cells are from the following species: humans, non-human mammals, poultry, plants, or microorganisms. Wherein, the non-human mammals include rodents (such as mice, rats, rabbits), cows, pigs, sheep, horses, dogs, cats, and non-human primates (such as monkeys).

In one embodiment of the present invention, the cell is selected from the group consisting of somatic cells, stem cells, germ cells, non-dividing cells or a combination thereof. Preferably, the cells are selected from the group consisting of kidney cells, epithelial cells, endothelial cells, nerve cells or a combination thereof.

In the present invention, when using the method for gene 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. 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 main advantages of the present invention include:

1) The editing window of the single-base editing system ABE is reduced, and the accuracy of single-base editing is greatly improved. When using the method of the present invention for gene 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 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, and it is distributed to both sides into a gradually decreasing trend. .

2) The point mutations generated by the single-base editing system ABE at the RNA level are almost eliminated, and the specificity of the single-base editing system ABE is greatly improved.

3) ABE7.10 ^F148A almost maintains the editing activity of ABE7.10, keeping the same activity in the target editing site.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory press, 1989), or according to the manufacturer’s The suggested conditions. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.

Unless otherwise specified, the materials and reagents used in the examples are all commercially available products.

Methods and materials

Transient transfection and sequencing

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 μg 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. In order to determine the efficiency of DNA base editing, 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. 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 editing analysis by RNA sequencing

Use Illumina Hiseq to perform high-throughput mRNA sequencing (RNA-seq) with an average coverage of 125x. FastQC (v0.11.3) and Trimmomatic (v0.36) are used for quality control. 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 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. In order to identify variants with high confidence, 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.

Any reliable variants found in wild-type 293T cells were considered SNPs and filtered from the GFP and base editor transfected group for off-target analysis. The edit rate is calculated as the number of mutation reads divided by the sequencing depth of each site. In order to analyze the predicted variation effect of each off-target, the variation effect predictor (VEP, v94) and GRCh38 database were used for variant annotation.

Single-cell full-length RNA-seq library construction

After FACS, a single human 293T cell was manually picked, lysed and cDNA synthesis was performed using the Smart-seq2 protocol. Then amplify and fragment single-cell cDNA as previously described (2,3). A sequencing library (New England Biolabs) was constructed, quality checked and sequenced on the Illumina HiSeq X-Ten platform (Novogene) with paired-end 150-bp reads.

Process single-cell 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.

Statistical Analysis

All values are shown as mean +/-SEM. Unpaired Student's t test (two-tailed) was used for comparison, and p<0.05 was considered statistically significant.

Example 1: Off-target RNA SNV detection for various single-base editing systems

In this example, in order to evaluate the off-target effect of gene editing at the RNA level, 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).

First, the use of targeted deep sequencing verified the high targeting efficiency of BE3 and ABE7.10 DNA editing in these 293T cells, and the results are shown in Figures 1B to 1E.

Next, 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.

The results are shown in Figures 1F to 1H and Figure 6. 742+/-113 (SEM, n=6) 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.

Interestingly, it was found in this example that 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.

Example 2: Characterization of off-target RNA SNV

In this example, off-target RNA SNV was characterized for each single-base editing system.

The results are shown in Figure 2 and Figure 7-12.

It is worth noting that almost 100% of 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.

Correspondingly, 95% of the mutations induced by ABE7.10 are A to G or U to C, which is consistent with the effect of ABE7.10 (Figures 2B and 2C and Figure 7).

From the results, it can also be noted that the GFP group also showed some deviations for A to G and U to C mutations (Figure 2C), which may be due to innate mutation preference.

In any two samples of the BE3- or ABE7.10-transfection group, an overlap of 27.7+/-3.6% or 51.0+/-3.3% was observed, respectively, and these overlapping SNVs were significant in genes with high expression Enriched (Figure 2D and Figure 8). However, no off-target sites overlap with predicted off-target mutations, and no similarity was observed between off-target and target sequences (Figure 2D and Figure 9).

Therefore, the off-target RNA and SNV induced by CBE and ABE are sgRNA-independent and caused by the overexpression of APOBEC1 and TadA-TadA*, respectively.

Interestingly, in this example, it was observed that ABE7.10 induced 56 and 12 non-synonymous RNA SNVs in oncogenes and tumor suppressor genes, many of which showed an editing rate higher than 40% and verified by Sanger sequencing , Which raises the concern about carcinogenic risk DNA base editing (Figure 2E, Figure 10 to 12).

Example 3: Single-cell RNA SNV analysis of cells transfected with single-base editing system

In this example, 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 .

The results are shown in Figure 3 and Figure 13-17.

On average, 10,932 RefSeq genes were detected in each single cell through approximately 6.07 million sequencing reads, and the results are shown in Figure 3B. Cells with high expression levels of the designated deaminase were selected for further analysis, and the results are shown in Figure 13. Also, severe RNA off-target and similar mutation patterns were observed in those cells expressing basic editing (Figures 3C to 3F and Figures 14 and 15).

Interestingly, the percentage of off-target sites shared by any BE3 or ABE7.10 editing cells (4.5+/-1.0%) is much lower than the cell population (40.8+/-3.7%), indicating that BE3- or ABE7.10 induced Off-target SNV is basically random and sgRNA independent (Figure 3G and 3H). It is worth noting that the editing rate of non-synonymous mutations detected in some oncogenes and tumor suppressor factors in single cells is higher than that observed from cell populations (Figure 3I, Figures 16 and 17).

Example 4: Elimination of off-target RNA by rational design of deaminase

In this example, in order to further explore experimental methods that may eliminate the off-target activity of base-edited RNA, the inventors studied the potential effects of destabilizing APOBEC1 and TadA on RNA binding.

Specifically, it was tested whether replacing APOBEC1 with hA3A can eliminate the RNA off-target activity of BE3 (Figure 4A).

The results are shown in Figure 4 and Figure 18-22.

In fact, 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).

In another method, 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).

In this example, for the modification of ABE, the inventors introduced D53G or F148A into TadA and TadA* of ABE7.10 (Figure 4A).

Interestingly, it was found that both ABE7.10 ^D53G and ABE7.10 ^F148A maintained high DNA targeting efficiency, and ABE7.10 ^F148A showed no RNA off-target effect at all. The results are shown in Figures 4D and 4E and Figure 18. In addition, the levels of SNV remaining in ABE7.10 ^F148A transfected cells were similar to those in cells transfected with GFP alone (Figures 19 to 21). In the present embodiment, it is also ^confirmed, ABE7.10 F148A DNA targeting activity on the other four sites with similar ABE7.10 (FIG. 4F).

It is particularly worth noting that in this embodiment, the editing window of ABE7.10 ^F148A is significantly reduced, and the results are shown in Figs. 4G and 22. This indicates that the accuracy of DNA base editing has improved.

Therefore, the engineered ABE7.10 ^{F148A in the} present invention has a larger application prospect.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. A mutant protein of adenine deaminase TadA, characterized in that the mutant protein is an unnatural protein, and the mutant protein occurs in one or more amino acids selected from the group consisting of the adenine deaminase TadA mutation:

   Phenylalanine (F) at position 147 and Phenylalanine (F) at position 148;

   Wherein, the 147th and 148th positions correspond to the 147th and 148th positions of the sequence shown in SEQ ID NO:1.
2. The mutant protein of claim 1, wherein the mutant protein has the activity of catalyzing the hydrolysis and deamination of adenine to form hypoxanthine.
3. The mutant protein of claim 1, wherein the adenine deaminase TadA includes TadA* enzyme and wild-type TadA enzyme.
4. A gene editing enzyme, characterized in that the structure of the gene editing enzyme is shown in formula I:

   Z1-L1-Z2-L2-Z3-L3-Z4 (I)

   among them,

   Z1 is the amino acid sequence of adenine deaminase TadA;

   Z2 is the amino acid sequence of TadA* enzyme;

   And said Z1 and/or Z2 is the amino acid sequence of the mutant protein according to claim 1;

   Z3 is the coding sequence of Cas9 nuclease;

   L1, L2 and L3 are each independently an optional connecting peptide sequence;

   Z4 is a non-or nuclear localization signal element (NLS);

   And each "-" is independently a peptide bond.
5. The gene-edited plum of claim 4, wherein the amino acid sequence of the gene-editing enzyme is shown in SEQ ID NO: 10.
6. A polynucleotide, wherein the polynucleotide encodes the gene editing enzyme according to claim 4.
7. A vector, characterized in that the vector contains the polynucleotide according to claim 6.
8. A host cell, characterized in that the host cell contains the vector according to claim 7, or the polynucleotide according to claim 6 is integrated into its genome.
9. A method for gene single-base directed editing, which is characterized in that it comprises the steps:

   (i) providing a cell and a first vector and a second vector, wherein the first vector contains an expression cassette for the gene editing enzyme according to claim 2, and the second vector contains an expression cassette for expressing sgRNA;

   (ii) Infecting the cell with the first vector and the second vector, thereby performing single-base site-directed editing in the cell.
10. A kit, characterized in that the kit includes:

    (a1) A first container, and a first vector located in the first container, the first vector containing the expression cassette of the gene editing enzyme according to claim 2.

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