CN115772512A - Adenine deaminase, adenine base editor containing adenine deaminase and application of adenine base editor - Google Patents

Abstract

The invention discloses an adenine deaminase, an adenine base editor containing the adenine deaminase and application of the adenine deaminase. The adenine deaminase is mutated at one or more amino acids at position 29, 84, 108 and 145 of the amino acid sequence comprising SEQ ID NO. 1. The adenine base editor comprises nuclease and the adenine deaminase, can effectively destroy the non-specific combination of the adenine deaminase and a substrate base, narrows an editing window to 1-2 bases, and maintains higher editing activity; structural pocket changes caused by mutation also make adenine deaminase unable to recognize cytosine as a substrate, and finally completely eliminate independent cytosine editing events existing in ABE; and the lower indel is kept, the safety is improved, the application of the compound in the aspects of accurate medical treatment, animal disease model making, crop genetic breeding and the like can be promoted, and the compound has great application value.

Description

Adenine deaminase, adenine base editor comprising it and application thereof

technical field

The invention belongs to the field of gene editing, and in particular relates to an adenine deaminase, an adenine base editor containing the same and an application thereof.

Background technique

The essence of human genetic diseases is due to gene mutations. About 60% of genetic diseases are caused by single base mutations. The traditional use of homologous recombination mediated by genome editing technology to correct such genetic diseases is very inefficient (0.1%-5 %). The single base editor (Base editor) derived from the CRISPR system is an emerging high-efficiency base editing technology in recent years. Because of its advantages such as no DNA double-strand breaks, no need for recombination templates, and efficient editing, it is widely used in basic research and clinical diseases. Therapeutics show great promise.

Classical base editors are mainly divided into cytosine base editors and adenine base editors. The former is composed of the Cas9 protein spCas9n from Streptococcus pyogenes with impaired activity, and cytosine base editors from rats. Composed of deaminase rAPOBEC1 and uracil glycosidase inhibitors, in which Cas9 protein uses NGG as PAM to recognize and specifically bind DNA, and then under the action of deaminase and DNA repair, it finally targets upstream of NGG (position 21-23). The C/G-T/A replacement is realized in the 20bp range of the sequence, and the editing window is mainly located at positions 4 to 8; the latter is the fusion of TadA (adenine deaminase) from bacteria and spCas9, which is used in directed evolution and protein engineering transformation With the help of technology, after 7 rounds of evolution, the adenine base editor ABE7.10, which can act on single-stranded DNA, is finally obtained. The active editing region is mainly located at positions 4-7. This system causes A/T-G/C in human cells The average editing efficiency is about 53%, which is much higher than the efficiency of base mutation mediated by homologous recombination. About 47% of human pathogenic point mutations are formed by the mutation of C·G to T·A, and the adenine base editor is expected to correct nearly half of the pathogenic point mutations, showing its role in mutation base modification and genetic disease. With great therapeutic potential, ABE has been widely used in animal model preparation and gene therapy.

In response to the low editing efficiency and narrow editing window of ABE7.10, various laboratories have carried out a lot of optimization and transformation work, using the strategy of replacing nuclear localization signals and codon optimization to obtain ABEmax. Compared with ABE7.10, A to G The highest editing efficiency was improved by 7.1 times. The CP-ABEmax series constructed by fusion of CP-Cas9 variants expanded the editing window from 4-7 positions to 4-12 positions, but the editing activity was still similar to ABEmax. In addition, in order to further expand the target of ABE ABEs with different PAM selectivities have also been developed, such as VQR-ABE(PAM:NGA), VRQR-ABE(PAM:NGA), SaCas9-ABE, (PAM:NNGRRT), SaKKH-ABE(PAM: NNNRRT), VRER-ABE(PAM:NGCG), xABEmax(PAM:NGN), NG-ABEmax(PAM:NG). With the help of molecular evolution technology, the newly reported ABE8e (Richter MF, et al. Phage-assisted evolution of an adenine base editor with improved cas domaincompatibility and activity. Nat Biotechnol, 2020, 38:883-891) and ABE8s (GaudelliNM, et al .Directed evolution of adenine base editors with increased activity and therapeutic application.Nat Biotechnol,2020,38:892-900), again significantly improved the efficiency of base editing, in which the activity of ABE8e increased by 590 times compared with ABE7.10, and at the same time The editing range is further expanded, covering 3 to 14 bits, which inevitably produces a serious "bystander effect", that is, all adenines in the window will be edited, and ABE8e and ABE8s cannot distinguish the target A when performing the editing function. and non-target A, so there is still a lack of high-precision and high-activity adenine base editors for the clinical application of precision medicine. At the same time, several research groups reported that ABE has harmful cytosine editing (Li S, et al. sites inside cas9 for adenine basedediting diversification and rna off-target elimination.Nat Commun,2020,11:5827;Kurt IC,et al.Crispr c-to-g base editors for inducing targeted DNAtransversions in human cells.Nat Biotechnol,2021, 39:41-46; Kim HS, et al. Adenine base editors catalyze cytosine conversions in human cells. Nat Biotechnol, 2019, 37: 1145-1148; Kim HS, et al. Adenine base editors catalyze cytosine conversions in human cells. Nat Biotechnol, 2019 , 37:1145-1148; Grunewald J, et al.Crispr DNA base editors with reduced rna off-target and self-editing activities.Nat Biotechnol,2019,37:1041-1048), which is bound to raise concerns about the safety of ABE applications.

At present, there is no report that can narrow the editing window of ABE to 1-2 bases. There is still a lack of effective base editors to achieve precise adenine editing, and the harmful cytosine editing produced by ABE has not been completely eliminated. Elimination, ABE security issues generated need to be resolved urgently.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the lack of ability to significantly reduce bystander adenine and cytosine editing in the prior art, and to provide an accurate, efficient, high-safety adenine deaminase, and an adenine base editor containing it. device and its application. The adenine base editor of the present invention can greatly reduce bystander adenine, almost completely eliminate bystander cytosine editing, and even narrow the editing window to 1-2 bases, and has extremely high safety.

The inventors predicted several key catalytic sites in TadA-8e through structural biology, on this basis, through amino acid substitutions, obtained single-point mutants based on TadA-8e, unexpectedly found that some single-point mutants greatly improved Destroyed the non-specific binding of adenine deaminase to substrate adenine while maintaining high editing activity; on the basis of single point mutants, double point mutants were further obtained through amino acid substitutions, and it was found that double point mutants could The editing window is narrowed to 1-2 bases, while maintaining high editing activity, and the structural pocket changes caused by the mutation also make it impossible for adenine deaminase to recognize cytosine as a substrate, and finally completely eliminate the presence of cytosine in ABE. Independent cytosine editing events, in addition still remain low indel.

The present invention solves the above-mentioned technical problems through the following technical solutions.

The first aspect of the present invention provides a kind of adenine deaminase, said adenine deaminase comprises the 29th, 84th, 108th and 145th positions of the amino acid sequence shown in SEQ ID NO:1 One or more amino acid mutations occur.

Preferably, the one or more amino acid mutations include:

Amino acid residue N at position 108 is mutated to Q; or,

Amino acid residue 145 L is mutated to T; or,

Amino acid residue L at position 145 is mutated to Q; or,

Amino acid residue F at position 84 is mutated to T; or,

Amino acid residue 84 F is mutated to T and amino acid residue 108 N is mutated to Q; or,

Amino acid residue N at position 108 is mutated to Q and amino acid residue 145 at L is mutated to T; or,

Amino acid residue N at position 108 is mutated to Q, and amino acid residue at position 29 P is mutated to M; or,

Amino acid residue N at position 108 was mutated to Q, and amino acid residue at position 29 P was mutated to W.

In the present invention, the amino acid sequence is shown in SEQ ID NO:1 and the nucleotide sequence of adenine deaminase is shown in SEQ ID NO:2.

In the present invention, the adenine deaminase also includes one or more amino acid mutations at other sites as shown in SEQ ID NO: 1, and the mutants formed have the same adenine deaminase as described in the first aspect. The same or similar function or biological activity as ammonia enzyme.

In some embodiments of the present invention, the adenine deaminase further includes a nuclear localization signal sequence; the nuclear localization signal sequence can be conventional in the art, for example, the nuclear localization signal sequence shown in SEQ ID NO:3.

The second aspect of the present invention provides an adenine base editor, which includes a nuclease and the adenine deaminase as described in the first aspect.

In some embodiments of the present invention, the nuclease is Cas protein and variants thereof;

In some preferred embodiments of the present invention, the Cas protein is spCas9 derived from Saccharomyces cerevisiae, SaCas9 derived from Staphylococcus aureus, LbCas12a derived from Lachnospiraceae bacteria or enAsCas12a derived from bacteria of the genus Amidococcus; Cas protein variants are VQR-spCas9, VRER-spCas9, spRY, spNG, SaCas9-KKH or SaCas9-NG.

In some embodiments of the invention, the adenine base editor significantly reduces bystander adenine editing and bystander cytosine editing.

In some embodiments of the present invention, the adenine base editor can greatly narrow the editing range, precisely edit 1-2 bases, and keep the occurrence of indel events low.

The third aspect of the present invention provides a fusion protein comprising the adenine deaminase as described in the first aspect.

In some embodiments of the present invention, the fusion protein further comprises a nuclease, and the definition of the nuclease is as described in the second aspect.

In the present invention, the sequence composition encoding the fusion protein can be promoter-adenine deaminase-nuclease-polyA, as long as it can provide the editing efficiency of A>G not lower than that of ABE8e; wherein, the promoter And the polyA can be conventional in the art.

The promoter can be CMV, or other types of spectrum promoters and tissue-specific promoters, such as CAG, PGK, EF1α; muscle-specific promoter Ctsk; liver-specific promoter Lp1, etc.

The polyA can be bovine growth hormone polyadenylation signal BGH polyA, or polyadenylation signal of other biological origin.

The sequence composition is, for example, CMV-adenine deaminase-Cas9n-BGH polyA.

The fourth aspect of the present invention provides an adenine base editing system, which includes: sgRNA and the adenine base editor as described in the second aspect.

In some embodiments of the present invention, the target sequence of the sgRNA is shown in the nucleotide sequence of SEQ ID NO: 4-15.

A fifth aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising the adenine deaminase as described in the first aspect, the adenine base editor as described in the second aspect, the adenine base editor as described in the third aspect The fusion protein or the adenine base editing system as described in the fourth aspect.

A sixth aspect of the present invention provides a non-therapeutic base editing method, the base editing method comprising:

Express the adenine deaminase as described in the first aspect, the adenine base editor as described in the second aspect, the fusion protein as described in the third aspect or the adenine as described in the fourth aspect in the target cell The purine base editing system, which causes base editing in the target cells, preferably further includes adding sgRNA, and the target sequence of the sgRNA is shown in the nucleotide sequence of SEQ ID NO: 4-15.

In some embodiments of the invention, the source of the target cells is an isolated cell line.

In some preferred embodiments of the present invention, the isolated cell line is 293T cells, HELA cells, U2OS cells, NIH3T3 cells or N2A cells.

In the present invention, the non-therapeutic purpose is to evaluate the adenine deaminase, adenine base editor, fusion protein or the adenine base described in the present invention by detecting the editing of target cells in the laboratory, for example. base editing system. Conversely, base editing can also be used to study the function of target cells.

In the present invention, the base editing method can also be used for therapeutic purposes. In the present invention, the treatment refers to the treatment of diseases in subjects such as humans, including inhibiting the occurrence or development of the diseases, alleviating the symptoms of the diseases or curing the diseases.

In the present invention, the target cells may be eukaryotic cells, prokaryotic cells, or archaic cells different from prokaryotic cells.

Preferably, the target cell can express the fusion protein as described in the third aspect.

More preferably, the target cells may be plant cells, human cells or animal cells.

The seventh aspect of the present invention provides the adenine deaminase as described in the first aspect, the adenine base editor as described in the second aspect, the fusion protein as described in the third aspect, or the fusion protein as described in the fourth aspect The application of the adenine base editing system in the preparation of base editing drugs or the preparation of gene therapy drugs.

The eighth aspect of the present invention provides the adenine deaminase as described in the first aspect, the adenine base editor as described in the second aspect, the fusion protein as described in the third aspect, or the fusion protein as described in the fourth aspect The application of the adenine base editing system in the construction of animal models and crop breeding.

The ninth aspect of the present invention provides the adenine deaminase as described in the first aspect, the adenine base editor as described in the second aspect, the fusion protein as described in the third aspect, or the fusion protein as described in the fourth aspect The application of the adenine base editing system in the preparation of base editing tools.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive progress effect of the present invention is:

The adenine base editor of the present invention can effectively destroy the non-specific binding between adenine deaminase and substrate bases, narrow the editing window to 1-2 bases, and maintain a high editing activity; The change in the structural pocket of ABE also makes it impossible for adenine deaminase to recognize cytosine as a substrate, and finally completely eliminates the independent cytosine editing event in ABE; and maintains a low indel, which improves safety and promotes its precise It has great application value in medical treatment, animal disease model making, crop genetic breeding and other applications.

Description of drawings

Figure 1 is the crystal structure of ABE8e bound to substrate DNA (PDB: 6VPC).

Figure 2 is a schematic diagram of the A>G base editing comparison results of 21 ABE8e mutants at the FANCF site1 site on 293T.

Figure 3 is a schematic diagram of the comparison results of A4 and C6 base editing achieved by 21 ABE8e mutants at the FANCF site1 site on 293T.

Figure 4 is a schematic diagram of the base editing comparison results of ABE8e and ABE8e-N108Q on 4 targets on 293T: A>G, C>G, C>T, and C>A.

Figure 5 is a schematic diagram of the comparison results of A>G base editing achieved by 19 ABE8e combined mutations at the ABE-site3 and ABE-site10 sites on 293T.

Figure 6 is a schematic diagram of the comparison results of A base and C base editing efficiency achieved by ABE9s and ABE9.1s at ABE-site10 and HEK-site7 sites on 293T.

Figure 7 shows the A>G editing efficiency of ABE9s, ABE9.1s, ABE9.2s, ABE9.3s and ABE9.4s on ABE-site16, ABE-site17, ABE-site13 and ABE-site8 endogenous targets on 293T Schematic diagram of the comparison results.

Figure 8 is a schematic diagram of the indel comparison results generated by ABE9s, ABE9.1s, ABE9.2s, ABE9.3s and ABE9.4s on ABE-site16, ABE-site17, ABE-site13 and ABE-site8 endogenous targets on 293T.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

Among the mutants used in the examples, the codons encoding the mutation sites are shown in Table 1.

Table 1 TadA single point mutation sequence

The targets and their sequences used in the examples are shown in Table 2.

Table 2 Targets and sequences used

target name Sequence (5'-3') SEQ ID NO FANCF site1 GGAATCCCTTCTGCAGCACC 4 EGFR-library-sg4 AAGATCAAAGTGCTGGGCTC 5 HBG-sg1 CTTGTCAAGGCTATTGGTCA 6 EMX1-sg2p GACATCGATGTCCTCCCCAT 7 HBG-sg8 CAGGACAAGGGAGGGAAGGA 8 ABE-site3 GTCAAGAAAGCAGAGACTGC 9 ABE-site10 GAACATAAAGAATAGAATGA 10 HEK-site7 GGAACACAAAGCATAGACTG 11 ABE-site16 GGGAATAAATCATAGAATCC 12 ABE-site17 GACAAAGAGGAAGAGAGACG 13 ABE-site13 GAAGATAGAGAATAGACTGC 14 ABE-site8 GTAAACAAAGCATAGACTGA 15

The identification primers of the targets used in the examples are shown in Table 3.

The identification primers of the targets used in Table 3

Among them: F is the forward primer, R is the reverse primer.

The seamless cloning kit used in the examples is Vazyme ClonExpress MultiS One StepCloning Kit, C113-01.

The HEK293T cells used in the examples are ATCC CRL-3216 cell lines.

The nucleotide sequence of the plasmid U6-sgRNA-EF1α-GFP used in the examples is shown in SEQ ID NO: 40, wherein the coding sequence of the sgRNA targeting the target sequence is represented by continuous N.

The service provider for sequencing in the examples is Suzhou Jinweizhi Biotechnology Co., Ltd.

Example 1

1.1 Plasmid design and construction

1.1.1 As shown in Figure 1, capture the crystal structure of ABE8e binding substrate DNA according to the cryo-electron microscope, and design 21 single point mutants of ABE8e (as shown in Table 1) according to the crystal structure, and design 1 at the same time The human endogenous test target FANCF site1 (shown in Table 2) was used for screening evaluation.

1.1.2 Synthesize 21 ABE8e single-point mutants according to the sequence in Table 1, use ABE8e as the vector, and then perform seamless cloning and assembly, that is, synthesize two oligos according to Table 2, add CACC to the positive strand, and add CACC to the reverse strand. AAAC, ligated to U6-sgRNA-EF1α-GFP digested with BbsI.

1.1.3 Sanger sequence the plasmids constructed in 1.1.1 and 1.1.2 to ensure that the sequences are completely correct.

1.2 Cell transfection

Day 1: Plating 24-well plates with 293T cells

(1) HEK293T cells were digested and inoculated into 96-well plates at 2×10 ⁵ cells/well.

Note: After recovery, the cells generally need to be subcultured twice before they can be used for transfection experiments.

Day 2: Transfection

(2) Observe the state of cells in each well.

Note: It is required that the cell density before transfection should be 70%-90%, and the state should be normal.

(3) The amount of plasmid transfection is as follows (using ABE8e as a control):

ABE8e single point mutant: U6-sgRNA-EF1α-GFP=750ng:250ng

Set n = 3 wells/group.

1.3 Genome extraction and amplicon library preparation

72h after transfection, the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Then, use the operating procedure of Hi-Tom Gene Editing Detection Kit (Novogene) to design corresponding identification primers (as shown in Table 3), that is, add a bridge sequence 5'-ggagtgagtacggtgtgc- at the 5' end of the forward primer 3' (SEQ ID NO: 41), add the bridge sequence 5'-gagttggatgctggatgg-3' (SEQ ID NO: 42) to the 5' end of the reverse primer to obtain a round of PCR product, and then use the round of PCR product as a template , Carry out two rounds of PCR to obtain the second round of PCR products, and then mix them together for gel cutting, recovery and purification, and then send them to the company for sequencing.

1.4 Analysis and statistics of deep sequencing results

Use the BE-analyzer website (http://www.rgenome.net/be-analyzer/#!) to analyze the deep sequencing results, that is, to count the ratios of A>G, C>T, C>G, C>A, and Indel, And use Graphpad Prism 9.1.0 for statistical mapping, as shown in Table 4 and Figures 2-4.

1.5 Result analysis

As shown in Table 4 and Figure 2, according to the Sanger results, among the 21 single point mutants of ABE8e, ABE8e-L145T, ABE8e-L145Q, ABE8e-N108Q, and ABE8e-F84T all significantly reduced bystander A3 editing while maintaining target Editing efficiency to base A4.

Table 4 Editing efficiency results of target FANCF site 1 (unit, %)

As shown in Figure 3, deep sequencing evaluated bystander C6 editing, bystander cytosine editing produced by ABE8e was 45.2% (C>G+C>T+C>A), while the above four single point mutants produced edits The efficiencies were only 5.07%, 3.67%, 2.44%, and 2.93%, respectively, and ABE8e-N108Q reduced the most harmful cytosine editing by 94.6%.

As shown in Figure 4, ABE8e-N108Q was selected to be verified again in four other endogenous targets (EGFR-library-sg4, HBG1-sg1, EMX1-sg2p, HBG-sg8). For the EGFR-library-sg4 target, ABE8e-N108Q reduced bystander cytosine editing from 13.7% to 3.13% for the HBG-sg1 target, and from 16.4% for the HBG-sg1 target to 5.17% for the EMX1-sg2P target , bystander cytosine editing decreased from 14.5% to 1.9%, and for the HBG-sg8 target, bystander cytosine editing decreased from 9.33% to 1.2%.

In summary, ABE8e-L145T, ABE8e-L145Q, ABE8e-N108Q and ABE8e-F84T all significantly reduced bystander adenine editing and bystander cytosine editing.

Example 2

This embodiment is designed to completely eliminate bystander cytosine editing and to achieve precise editing of a single A>G.

2.1 Plasmid design and construction

2.1.1 Based on the results of single-point mutation screening in Example 1, and again according to the crystal structure of ABE8e, amino acid sites that potentially affect the structure of the substrate were subjected to combinatorial mutation synthesis for seamless cloning assembly. At the same time, two poly A-rich endogenous test targets, ABE site10 and ABE site3, were designed for testing (as shown in Table 2), and the construction method was the same as

1.1.2.

2.1.2 Sanger sequenced the plasmid constructed in 2.1.1 to ensure that it is completely correct.

2.2 Cell transfection

Day 1: Plating 24-well plates with 293T cells

(1) HEK293T cells were digested and inoculated into 96-well plates at 2×10 ⁵ cells/well.

Note: After recovery, the cells generally need to be subcultured twice before they can be used for transfection experiments.

Day 2: Transfection

(2) Observe the state of cells in each well.

Note: It is required that the cell density before transfection should be 70%-90%, and the state should be normal.

(3) The amount of plasmid transfection is as follows (using ABE8e as a control)

Newly constructed plasmid in 2.1: U6-sgRNA-EF1α-GFP=750ng:250ng

Set n = 3 wells/group.

2.3 Genome extraction and amplicon library preparation

72h after transfection, the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Afterwards, use the operation process of the Hitom kit to design corresponding identification primers (as shown in Table 3), that is, add the bridging sequence shown in SEQ ID NO:38 at the 5' end of the forward primer, and the 5' end of the reverse primer Add the bridging sequence shown in SEQ ID NO:39 to the end to obtain a round of PCR product, and then use the round of PCR product as a template to perform a second round of PCR to obtain a second round of PCR product, which is then mixed together for recovery and purification by cutting the gel Sent to the company for sequencing.

2.4 Analysis and statistics of deep sequencing results

Use the BE-analyzer website (http://www.rgenome.net/be-analyzer/#!) to analyze the deep sequencing results, that is, to count the ratio of A>G, C>T, C>G, C>A, Indel, And use Graphpad Prism 9.1.0 for statistical graphing.

2.5 Result analysis

As shown in Figure 5, according to the Sanger results, among the 19 combined mutations, N108Q-L145T, N108Q-P29M, N108Q-P29W, and N108Q-F84T showed a very narrow editing window, and the targeting range was about 1 to 2 bases . For the ABEsite3 target, the editing range of ABE8e is ~5 bases, the reported F148A mutation with the potential to narrow the window also has an editing range of ~4 bases, the editing window of ABE8e-N108Q is ~3 bases, and the four This combination mutation only edits 1 base, and edits A5 efficiently and accurately. Also for the ABE site10 target, ABE8e covers ~7 bases, ABE8e-F148A targets ~4 bases, for the single point mutation ABE8e-N108Q, the editing range is still ~3 bases, N108Q-L145T and The editing range of N108Q-P29M is ~2 bases, and it also efficiently catalyzes A>G at the fifth position, while N108Q-P29W and N108Q-F84T precisely edit one base A5, and the editing activity is only partially reduced. For ease of description, ABE8e-N108Q was named ABE9s, and N108Q-L145T, N108Q-P29M, N108Q-P29W, and N108Q-F84T were named ABE9.1s, ABE9.2s, ABE9.3s, and ABE9.4s in turn. The editing window of ABE9.1s, ABE9.2s, ABE9.3s and ABE9.4s is about 1-2 bases, and the selectivity of adenine editing is strict in order.

As shown in Figure 6, taking ABE9.1s as an example, taking ABE site10 and the newly designed endogenous target HEK site7 as evaluation targets, the bystander cytosine editing characteristics of combined mutations were evaluated again. The results showed that compared with ABE9s, ABE9.1s completely eliminated harmful cytosine editing, narrowed the window to 1-2 bases, and preferentially edited A5/A6 bases.

In summary, the combined mutations N108Q-L145T, N108Q-P29M, N108Q-P29W, and N108Q-F84T of ABE8e can precisely edit 1-2 bases while completely eliminating harmful cytosine editing.

Example 3

This embodiment is designed to compare the working characteristics of ABE9.1s, ABE9.2s, ABE9.3s and ABE9.4s.

3.1 Plasmid design and construction

3.1.1 Using ABE8e and ABE9s as controls, design 4 additional targets ABE-site16, ABE-site17, ABE-site13 and ABE-site8 (as shown in Table 2) for evaluation.

3.1.2 Sanger sequenced the plasmid constructed in 3.1.1 to ensure that it is completely correct.

3.2 Cell transfection

Day 1: Plating 24-well plates with 293T cells

(1) HEK293T cells were digested and inoculated into 96-well plates at 2×10 ⁵ cells/well.

Note: After recovery, the cells generally need to be subcultured twice before they can be used for transfection experiments.

Day 2: Transfection

(2) Observe the state of cells in each well.

Note: It is required that the cell density before transfection should be 70%-90%, and the state should be normal.

(3) The amount of plasmid transfection is as follows (using ABE8e as a control):

Newly constructed plasmid in 3.1: U6-sgRNA-EF1α-GFP=750ng:250ng

Set n = 3 wells/group.

3.3 Genome extraction and amplicon library preparation

72h after transfection, the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Afterwards, use the operation process of the Hitom kit to design corresponding identification primers (as shown in Table 3), that is, add the bridging sequence shown in SEQ ID NO:38 at the 5' end of the forward primer, and the 5' end of the reverse primer Add the bridging sequence shown in SEQ ID NO:39 to the end to obtain a round of PCR product, and then use the round of PCR product as a template to perform a second round of PCR to obtain a second round of PCR product, which is then mixed together for recovery and purification by cutting the gel Then send it to the company for sequencing.

3.4 Analysis and statistics of deep sequencing results

Use the BE-analyzer website (http://www.rgenome.net/be-analyzer/#!) to analyze the deep sequencing results, that is, to count the ratio of A>G, C>T, C>G, C>A, Indel, And use Graphpad Prism 9.1.0 for statistical graphing.

3.5 Result analysis

As shown in Figure 7, at ABE site16, the corresponding ABE8e editing window is ~5 bases, ABE9s can cover ~4 bases, ABE9.1s, ABE9.2s, ABE9.3s, ABE9.4s only edit 1 to 2 bases; at ABE site13 and ABEsite13, the editing range of ABE8e is ~5 bases, and the coverage of ABE9s is 3 to 4 bases, while ABE9.1s, ABE9.2s, ABE9.3s and ABE9 In .4s, except for ABE9.1s with slight A7 or A4 editing, the other three variants only edited one base. For the ABE site17 site, the editing range of ABE8e is ~6 bases, the editing range of ABE9s is ~3 bases, ABE9.1s and ABE9.2s mainly edit 2 bases (A5/A6), ABE9.3s and ABE9 .4s still edits single bases precisely.

In summary, ABE9s can slightly narrow the editing range, while ABE9.1s, ABE9.2s, ABE9.3s, and ABE9.4s can precisely edit 1 to 2 bases, while maintaining a low occurrence of indel events (as shown in Figure 8 ).

SEQUENCE LISTING

<110> East China Normal University

Shanghai Bangyao Biotechnology Co., Ltd.

<120> Adenine deaminase, adenine base editor comprising same and application thereof

<130> P21016504C

<160> 42

<170> PatentIn version 3.5

<210> 1

<211> 167

<212> PRT

<213> Artificial Sequence

<220>

<223> Tad A

<400> 1

Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu

1 5 10 15

Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala

20 25 30

Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala

35 40 45

Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg

50 55 60

Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu

65 70 75 80

Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His

85 90 95

Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ser Lys Arg Gly

100 105 110

Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met Asn His

115 120 125

Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu

130 135 140

Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys

145 150 155 160

Lys Ala Gln Ser Ser Ile Asn

165

<210> 2

<211> 501

<212>DNA

<213> Artificial Sequence

<220>

<223> Tad A

<400> 2

atgtctgagg tggagttttc ccacgagtac tggatgagac atgccctgac cctggccaag 60

agggcacggg atgagaggga ggtgcctgtg ggagccgtgc tggtgctgaa caatagagtg 120

atcggcgagg gctggaacag agccatcggc ctgcacgacc caacagccca tgccgaaatt 180

atggccctga gacagggcgg cctggtcatg cagaactaca gactgattga cgccaccctg 240

tacgtgacat tcgagccttg cgtgatgtgc gccggcgcca tgatccactc taggatcggc 300

cgcgtggtgt ttggcgtgag gaactcaaaa agaggcgccg caggctccct gatgaacgtg 360

ctgaactacc ccggcatgaa tcaccgcgtc gaaattaccg agggaatcct ggcagatgaa 420

tgtgccgccc tgctgtgcga tttctatcgg atgcctagac aggtgttcaa tgctcagaag 480

aaggcccaga gctccatcaa c 501

<210> 3

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> Nuclear localization signal sequence

<400> 3

Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg

1 5 10 15

Lys Val

<210> 4

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> FANCF site1

<400> 4

ggaatccctt ctgcagcacc 20

<210> 5

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> EGFR-library-sg4

<400> 5

aagatcaaag tgctgggctc 20

<210> 6

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> HBG-sg1

<400> 6

cttgtcaagg ctattggtca 20

<210> 7

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> EMX1-sg2p

<400> 7

gacatcgatg tcctccccat 20

<210> 8

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> HBG-sg8

<400> 8

caggacaagggagggaagga 20

<210> 9

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site3

<400> 9

gtcaagaaag cagagactgc 20

<210> 10

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site10

<400> 10

gaacataaag aatagaatga 20

<210> 11

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> HEK-site7

<400> 11

ggaacacaaa gcatagactg 20

<210> 12

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site16

<400> 12

gggaataaat catagaatcc 20

<210> 13

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site17

<400> 13

gacaaagagg aagagagacg 20

<210> 14

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site13

<400> 14

gaagatagag aatatagactgc 20

<210> 15

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site8

<400> 15

gtaaacaaag catagactga 20

<210> 16

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223> FANCF site1-F

<400> 16

ggagtgagta cggtgtgcaa ggaacacgga taaagacgct ggg 43

<210> 17

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223> FANCF site1-R

<400> 17

gagttggatg ctggatggta ggtagtgctt gagaccgcca gaa 43

<210> 18

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223> EGFR-library-sg4-F

<400> 18

ggagtgagta cggtgtgcct tgtggagcct cttacaccca gtg 43

<210> 19

<211> 41

<212>DNA

<213> Artificial Sequence

<220>

<223> EGFR-library-sg4-R

<400> 19

gagttggatg ctggatggct ccccaccaga ccatgagagg c 41

<210> 20

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223> HBG-sg1-F

<400> 20

ggagtgagta cggtgtgctg gaatgactga atcggaacaa ggc 43

<210> 21

<211> 44

<212>DNA

<213> Artificial Sequence

<220>

<223> HBG-sg1-R

<400> 21

gagttggatg ctggatggct ggcctcactg gatactctaa gact 44

<210> 22

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223>EMX1-sg2p-F

<400> 22

ggagtgagta cggtgtgcgt ggttccagaa ccggaggaca aag 43

<210> 23

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223> EMX1-sg2p-R

<400> 23

gagttggatg ctggatgggt ttgtggttgc ccaccctagt cat 43

<210> 24

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223> HBG-sg8-F

<400> 24

ggagtgagta cggtgtgctg gggcaaggtg aatgtggaag atg 43

<210> 25

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223> HBG-sg8-R

<400> 25

gagttggatg ctggatggaa cctctgggtc catgggtaga caa 43

<210> 26

<211> 44

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site3-F

<400> 26

ggagtgagta cggtgtgctg tcttcccttt cccttttcct cacc 44

<210> 27

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site3-R

<400> 27

gagttggatg ctggatggaa ttgaggctca gaggagatgt gcc 43

<210> 28

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site10-F

<400> 28

ggagtgagta cggtgtgcta cattaaccat ccccacatta tcc 43

<210> 29

<211> 45

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site10-R

<400> 29

ggttggatg ctggatggag ggaactagat gttatgttta ggtga 45

<210> 30

<211> 44

<212>DNA

<213> Artificial Sequence

<220>

<223> HEK-site7-F

<400> 30

ggagtgagta cggtgtgctg aatggattcc ttggaaacaa tgat 44

<210> 31

<211> 41

<212>DNA

<213> Artificial Sequence

<220>

<223> HEK-site7-R

<400> 31

gagttggatg ctggatggtg tcaaactgtg cgtatgacat c 41

<210> 32

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site16-F

<400> 32

ggagtgagta cggtgtgcta caattctgac cccatgcacc ctc 43

<210> 33

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site16-R

<400> 33

gagttggatg ctggatggat gccagatacc agcaatccag caa 43

<210> 34

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site17-F

<400> 34

ggagtgagta cggtgtgcct caagcctgat tccaaggaga ttg 43

<210> 35

<211> 39

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site17-R

<400> 35

gagttggatg ctggatggtc cctcctctgc gtgaatttg 39

<210> 36

<211> 44

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site13-F

<400> 36

ggagtgagta cggtgtgcca tcaatcaact tctctttctc tccc 44

<210> 37

<211> 44

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site13-R

<400> 37

gagttggatg ctggatggat atcacttcag cccaggagta taac 44

<210> 38

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site8-F

<400> 38

ggagtgagta cggtgtgcct gctgccgtgg gagacaattc ata 43

<210> 39

<211> 43

<212>DNA

<213> Artificial Sequence

<220>

<223> ABE-site8-R

<400> 39

gagttggatg ctggatggag ctgttgcatg aggaaaggga cta 43

<210> 40

<211> 2340

<212>DNA

<213> Artificial Sequence

<220>

<223> U6-sgRNA-EF1α-GFP

<220>

<221> misc_feature

<222> (250)..(269)

<223> n is a, c, g, or t

<400> 40

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccn nnnnnnnnnn nnnnnnnnng ttttagagct agaaatagca agttaaaata 300

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttaggcctga 360

attctgcaga tatccatcac actggcggct ccggtgcccg tcagtggggca gagcgcacat 420

cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa 480

ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg 540

gtggggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt 600

ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg 660

gttatggccc ttgcgtgcct tgaattactt ccactggctg cagtacgtga ttcttgatcc 720

cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc 780

gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg 840

gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccattaaa atttttgatg 900

acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca 960

cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac 1020

atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca 1080

agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc 1140

ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc 1200

tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc 1260

cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta 1320

ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg 1380

ttggggggag gggttttatg cgatggagtt tccccaacact gagtgggtgg agactgaagt 1440

taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc 1500

ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc 1560

gtgaaatacg actcactata gggagaccca agctggctag ttaagcttgg taccgccacc 1620

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 1680

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 1740

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 1800

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 1860

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 1920

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 1980

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 2040

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 2100

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 2160

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 2220

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 2280

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 2340

<210> 41

<211> 18

<212>DNA

<213> Artificial Sequence

<220>

<223> Forward primer end bridge sequence

<400> 41

ggagtgagta cggtgtgc 18

<210> 42

<211> 18

<212>DNA

<213> Artificial Sequence

<220>

<223> Reverse primer end bridging sequence

<400> 42

gagttggatg ctggatgg 18

Claims (10)

1. An adenine deaminase comprising one or more amino acid mutations at position 29, 84, 108 and 145 of the amino acid sequence as set forth in SEQ ID NO 1.

2. The adenine deaminase of claim 1, wherein said one or more amino acid mutations comprise:

the 108 th amino acid residue N is mutated into Q; or the like, or, alternatively,

the 145 th amino acid residue L is mutated into T; or the like, or a combination thereof,

the 145 th amino acid residue L is mutated into Q; or the like, or a combination thereof,

the 84 th amino acid residue F is mutated into T; or the like, or a combination thereof,

the 84 th amino acid residue F is mutated into T, and the 108 th amino acid residue N is mutated into Q; or the like, or, alternatively,

the 108 th amino acid residue N is mutated into Q, and the 145 th amino acid residue L is mutated into T; or the like, or a combination thereof,

the 108 th amino acid residue N is mutated into Q, and the 29 th amino acid residue P is mutated into M; or the like, or, alternatively,

the 108 th amino acid residue N is mutated into Q, and the 29 th amino acid residue P is mutated into W;

preferably, the adenine deaminase further comprises a nuclear localization signal sequence; the nuclear localization signal sequence is preferably shown as SEQ ID NO. 3.

3. An adenine base editor comprising a nuclease and an adenine deaminase of claim 1 or 2;

preferably, the nuclease is a Cas protein and variants thereof;

more preferably, the Cas protein is saccharomyces cerevisiae-derived spCas9, staphylococcus aureus-derived SaCas9, laccas 12a derived from lachnospiraceae bacteria, or enacas 12a derived from acidaminococcus bacteria; the Cas protein variant is VQR-spCas9, VRER-spCas9, sprY, spNG, saCas9-KKH or SaCas9-NG.

4. A fusion protein comprising the adenine deaminase of claim 1 or 2;

preferably, the fusion protein further comprises a nuclease, the nuclease is the nuclease in the adenine base editor of claim 3.

5. An adenine base editing system comprising: sgRNA and the adenine base editor of claim 3;

preferably, the target sequence of the sgRNA is shown as the nucleotide sequence of SEQ ID NO. 4-15.

6. A pharmaceutical composition comprising the adenine deaminase of claim 1 or 2, the adenine base editor of claim 3, the fusion protein of claim 4 or the adenine base editing system of claim 5.

7. A method of base editing for non-therapeutic purposes, the method comprising:

expressing the adenine deaminase of claim 1 or 2, the adenine base editor of claim 3, the fusion protein of claim 4, or the adenine base editing system of claim 5 in a target cell such that base editing occurs in said target cell;

preferably, the source of the target cells is an isolated cell line;

more preferably, the isolated cell line is a 293T cell, a HELA cell, a U2OS cell, an NIH3T3 cell or an N2A cell.

8. Use of the adenine deaminase of claim 1 or 2, the adenine base editor of claim 3, the fusion protein of claim 4 or the adenine base editing system of claim 5 in the manufacture of a medicament for base editing or in the manufacture of a medicament for gene therapy.

9. Use of the adenine deaminase of claim 1 or 2, the adenine base editor of claim 3, the fusion protein of claim 4 or the adenine base editing system of claim 5 in the construction of animal models and crop breeding.

10. Use of an adenine deaminase according to claim 1 or 2, an adenine base editor according to claim 3, a fusion protein according to claim 4 or an adenine base editing system according to claim 5 for the preparation of a base editing tool.

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