KR20220077054A - Adenine base editors with enhanced thymine-cytosine sequence-specific cytosine editing activity and use thereof - Google Patents

Abstract

The present invention provides a thymine-cytosine sequence-specific cytosine sequence-specific cytosine-correcting activity, adenine base editing gene scissors, and a thymine-cytosine sequence-specific cytosine sequence-specific cytosine base editing composition comprising the gene scissors and guide RNA, cytosine base It relates to a calibration method and a kit for cytosine base calibration. The adenine base editing gene scissors with enhanced T-C sequence-specific cytosine editing activity according to the present invention was produced by introducing the P48R mutation to an adenosine deamination enzyme, and has a more sophisticated operating range than the conventional cytosine base editing gene scissors. Since cytosine correction occurs only when thymine is located immediately before cytosine, sophisticated correction is possible even when there are several cytosines within the range. By adding UGI, cytosine can be substituted with thymine, Cytosine may be substituted with guanine. Therefore, the composition for cytosine base editing comprising cytosine base editing gene scissors and sgRNA according to the present invention is a gene therapy field or a new crop that needs to accurately correct only cytosine in all living things including humans, plants, and bacteria It will be useful in the field of development, etc.

Description

Adenine base editors with enhanced thymine-cytosine sequence-specific cytosine editing activity and use thereof {Adenine base editors with enhanced thymine-cytosine sequence-specific cytosine editing activity and use thereof}

The present invention relates to a thymine-cytosine sequence-specific cytosine sequence-specific cytosine editing activity, adenine nucleotide correction gene scissors, a thymine-cytosine sequence-specific cytosine sequence-specific cytosine nucleotide correction composition comprising the gene scissors and guide RNA, cytosine nucleotide correction It relates to a method and a kit for cytosine base correction.

Adenine base editors (ABEs) are effective gene editing tools that can convert A/T pairs to G/C pairs without generating DNA double-strand breaks (DSBs) or requiring donor DNA templates. . These technologies are being used to correct bases not only at the cellular level but also in plants or animals, and verification and development are being actively carried out so that they can be used for gene therapy. The early practical version of the ABE (ABE7.10) consisted of three fused elements. Specifically, it consists of a pair of partially inactive Cas nuclease (Cas nickase or nCas) and adenosine deaminases (wild-type tRNA-specific adenosine deaminase, TadA from Escherichia coli (wtTadA) Enzyme, and engineered TadA (eTadA), which is TadA7.10 evolved to work on DNA instead of RNA). However, in recent adenine sequencing, genome-wide guide RNA (sgRNA)-dependent off-target DNA editing, transcript-wide sgRNA-independent off-target RNA editing, and ABE-mediated cytosine deamination effect at off-target sites, etc. This has been reported The first off-target effect is caused by the imperfect target specificity of Cas nuclease, and the other two off-target effects are due to the DNA/RNA-binding properties of adenosine deaminase.

To date, several research groups have developed a new version of ABE that introduces additional mutations to reduce off-target effects on RNA. For example, it was reported that the addition of the F148A mutation in both wtTadA and TadA7.10 reduced the random RNA deamination activity. Another group independently reported that wtTadA of ABE7.10 mainly affected RNA deamination activity, whereas it was found that the absence of wtTadA did not affect DNA editing activity. We therefore inactivated wtTadA by introducing the E59A mutation, and found that an additional mutation in TadA7.10 (V106W) reduced the off-target effect on RNA without reducing DNA targeting activity. Another group completely eliminated wtTadA and added several mutations to TadA7.10 (K20A/R21A or V82G) to reduce RNA off-target effects (Nat Biotechnol 37, 1041-1048 (2019)). However, studies have been conducted mainly focusing on reducing ABE-mediated RNA deamination activity in many research groups as described above.

On the other hand, cytosine base editors (CBEs) are effective gene editing tools known to be capable of replacing cytosine with thymine without generating DNA double-strand breaks (DSBs) or using a donor DNA template. However, since the cytosine base editing gene has a wide operating range (position 3 to 9), there is a problem that correction occurs even at unwanted bases, and there is a problem that is not suitable for substituting cytosine with a base other than thymine.

Accordingly, in the present invention, a key mutation capable of improving cytosine catalysis in adenosine deamination enzyme (TadA) was discovered and a gene scissors capable of efficient and sophisticated cytosine correction was developed.

The present inventors rationally design and test dozens of TadA variants in order to develop gene scissors for more efficient and sophisticated cytosine base correction, and have identified mutations that improve cytosine correction activity. Based on this, the present invention was completed. did

Accordingly, an object of the present invention is to provide an adenine base editor with enhanced thymine-cytosine (T-C) sequence-specific cytosine editing activity.

In addition, the present invention is the adenine base correction gene scissors; and guide RNA (single guide RNA; sgRNA), including, thymine-cytosine (T-C) sequence-specific cytosine base correction composition for another object.

Another object of the present invention is to provide a thymine-cytosine (TC) sequence-specific cytosine base correction method comprising the step of contacting the composition with a target sequence in vitro .

In addition, it is another object of the present invention to provide a kit for thymine-cytosine (T-C) sequence-specific cytosine base correction comprising the composition.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention is adenosine deaminase (Adenosine deaminase) variant comprising a P48R mutation; and Cas9 (CRISPR associated protein 9) protein fused, thymine-cytosine (T-C) provides an adenine base editor with enhanced sequence-specific cytosine editing activity (Adenine Base Editor).

In one embodiment of the present invention, the adenosine deamination enzyme may be TadA7.10.

In another embodiment of the present invention, the gene scissors may be a mutation introduced into ABEmax.

In another embodiment of the present invention, the gene scissors may be one in which one or more uracil DNA glycosylase (UGI) is additionally linked.

In addition, the present invention is the adenine base correction gene scissors; and

It provides a composition for thymine-cytosine (T-C) sequence-specific cytosine base correction, including a guide RNA (single guide RNA; sgRNA).

In one embodiment of the present invention, the cytosine may be a cytosine (C) located at the 5th to 7th positions from the 5' end of the target sequence.

In another embodiment of the present invention, the composition may be to substitute thymine (T) or guanine (G) for cytosine (C) positioned immediately after thymine (T) on the target sequence depending on the presence or absence of UGI.

In addition, the present invention provides a thymine-cytosine (TC) sequence-specific cytosine base correction method comprising the step of contacting the composition with a target sequence in vitro .

In addition, the present invention, comprising the composition, thymine-cytosine (T-C) provides a kit for sequence-specific cytosine base correction.

According to the present invention, the T-C sequence-specific cytosine correction activity is enhanced, and the adenine base editing gene is produced by introducing the P48R mutation to an adenosine deaminase, and a more sophisticated operating range than the conventional cytosine base editing gene scissors. Since cytosine correction occurs only when thymine is located immediately before cytosine, sophisticated correction is possible even when there are several cytosines within the range. By adding UGI, cytosine can be substituted with thymine, In some cases, cytosine may be substituted with guanine. Therefore, the composition for cytosine base editing comprising cytosine base editing gene scissors and sgRNA according to the present invention is a gene therapy field or a new crop that needs to accurately correct only cytosine in all living things including humans, plants, and bacteria It will be useful in the field of development, etc.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 pictorially depicts a schematic diagram of targeted adenine and cytosine correction mediated by the catalytic activity of ABE.
Figure 2 is a graph showing the nucleotide correction efficiency of each of A4 and C6 of ABE at FANCF and RNF2 positions and adenosine deaminase (left) included in various adenine base correction gene scissors (right).
3 is a result of analyzing the amino acid sequence of adenosine deamination enzyme (TadA) and its structure. , Fig. 3b shows the results of superimposing the apo structure derived from E. coli (green; PDB code 1Z3A) on the RNA-bound saTadA single structure (pink and gray; PDB code 2B3J).
4 is a heat map result analyzing the adenine and cytosine correction efficiency induced by 33 ABE variants at the FANCF and RNF2 positions (left) and the accuracy value obtained by dividing the adenine correction efficiency by the cytosine correction efficiency as a graph. (right).
Figure 5 shows the conventionally known cytosine base correction gene scissors (CBE and CBE (ΔUGI)), adenine base correction gene scissors (ABEmax and ABEmax-UGI) and adenine base correction gene scissors (ABE-P48R and P48R mutations) This is a diagram showing the components of ABE-P48R-UGI).
6 is a heat map showing the substitution of bases for the target site of the CSRNP3 gene by performing high-throughput sequencing after transfecting cells with each of the nucleotide-correcting gene scissors of FIG. 5 .
Figure 7 is a high-throughput sequencing analysis after transfecting each of the base-correcting gene scissors of Figure 5 into cells, and the cytosine of each TC motif of the four endogenous genes ( FANCF , RNF2 , ABLIM3 and CSRNP3 ) is substituted with a certain tendency. This is the result of analyzing and showing whether or not
Figure 8 is adenine base editing scissors (ABEmax and ABEmax-UGI) and P48R mutation introduced adenine base editing scissors (ABE-P48R and ABE-P48R-UGI) cytosine correction of TC motif after transfection into cells It is the result of confirming the effect according to the position of cytosine.
9 is a motif of the cytosine correction effect after transfecting cells with adenine base editing scissors (ABEmax and ABEmax-UGI) and adenine base editing scissors (ABE-P48R and ABE-P48R-UGI) introduced with P48R mutations. It is the result of checking according to the type.
10 is a diagram showing the number of diseases caused by substitution of thymine with cytosine and diseases caused by substitution of guanine with cytosine using the ClinVar database.
Figure 11 shows the cytosine correction efficiency after transfecting ABE-P48R-UGI and CBE (AncBE4max) of the present invention into a cell line that mimics the genetic mutation of a missense mutation-related disease of the TUBB6 gene in which the TT sequence is mutated to TC. It is the result of comparing the possibility of treatment by measuring.
12 is a cell line mimicking the genetic mutation of a non-toxic and toxic goitre disease related to a missense mutation of the TPO gene in which the TG sequence is mutated to TC, after transfection of ABE-P48R and CBE (AncBE4max) of the present invention. It is the result of comparing the treatment potential by measuring the correction efficiency.

Hereinafter, the present invention will be described in detail.

The present inventors have identified a major mutation that significantly reduced adenine base editing activity and enhanced cytosine editing activity in adenine base editing gene scissors, and introduced the mutation into adenosine deaminase to specifically cytosine-cytosine sequence. Adenine base-correcting gene scissors with improved renal-correcting activity were prepared.

The present invention provides an adenosine deaminase variant comprising a P48R mutation; and

Provided is an adenine base editor (Adenine Base Editor) in which Cas9 (CRISPR associated protein 9) protein is fused, thymine-cytosine (T-C) sequence-specific cytosine editing activity is enhanced.

As used in the present invention, the term "Base Editors (BEs)" is a single base editing means, more specifically, adenosine deaminase or cytosine deamination enzyme Cas9 nickase (nickase) It is constructed by fusion to the N-terminus, and is named Adenine Base Editors (ABEs) and Cytosine Base Editors (CBEs), respectively. The BEs do not cause double-strand breaks, while ABEs correct adenine to guanine at a specific site, and CBEs correct cytosine to thymine at a specific site.

As used herein, the term "Adenine Base Editors (ABEs)" refers to any naturally occurring deamination enzyme (ecTadA) and / or adenosine deamination enzyme variant ( It is constructed by fusing ecTadA*) to the N-terminus of Cas9 nickase, and the types of ABEs include ABE6.3, ABE7.8, ABE7.9, ABE 7.10 depending on the type or mutation of the adenosine deaminase. , ABEmax, ABEmax-m, SECURE-ABE, ABE8e, ABE8e-V106W, ABE8.17-m, etc., but are not limited thereto, and may be referred to as “ABEs”.

In the present invention, the adenine base correction gene scissors may have improved specificity or accuracy for adenine base correction, and have the characteristics of significantly increasing the cytosine editing activity specifically for the thymine-cytosine sequence. Preferably, the mutation may be induced in the ABEmax version, but is not limited thereto.

As used herein, the term "adenosine deaminase" is an enzyme that removes an amino group from adenine and is involved in the production of hypoxanthine. It is reported that the enzyme is rarely found in higher animals, but is present in small amounts in the muscles of cows, milk, and blood of mice, and exists in large amounts in the intestines of crayfish and insects. The adenosine deaminase may include a naturally occurring adenosine deaminase such as ecTadA or a variant of an adenosine deaminase such as a mutant (ecTadA*) of ecTadA, preferably the variant is TadA7.10, TadA8e, TadA8s, TadA8.20 or TadA8.17 may be included, and in the present invention, TadA7.10 is used, but is not limited thereto.

As used herein, the term “Cas9 (CRISPR associated protein 9) protein” is a protein that plays an important role in the immunological defense of specific bacteria against DNA viruses and is widely used in genetic engineering applications. It can be applied to modify the genome of a cell because it cuts the Specifically, CRISPR-Cas9 recognizes, cuts, and edits a specific nucleotide sequence to be used as a third-generation gene scissors, and inserts a specific gene into the target site of the genome or stops the activity of a specific gene simply, quickly and efficiently It is useful to carry out Cas9 protein or gene information may be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI), but is not limited thereto. In addition, according to the purpose of the Cas9 protein, a person skilled in the art can properly link additional domains. In the present invention, the Cas9 protein may include all variants of Cas9 as long as it has the function of a nuclease for gene editing as well as wild-type Cas9.

In addition, the Cas9 protein in the present invention is not limited in its origin, as a non-limiting example Streptococcus pyogenes , Francisella novicida ( Francisella novicida ), Streptococcus thermophilus ( Streptococcus thermophilus ), Legionella It may be derived from Pneumophila ( Legionella pneumophila ), Listeria innocua , or Streptococcus mutans , but preferably from Streptococcus pyogenes ).

In the present invention, the gene scissors may be one in which one or more uracil DNA glycosylase (UGI) is additionally linked, and preferably, two UGIs are additionally linked. The UGI, also referred to as UNG or UDG, serves to hydrolyze the N-glycosylic bond between the deoxyribose sugar of DNA including uracil and the uracil base to create a depilimidine site. It is known that the enzyme hydrolyzes single-stranded DNA and double-stranded DNA including uracil, but does not act on RNA.

The present inventors confirmed that the T-C sequence-specific cytosine editing activity was improved in the improved adenine base editing gene scissors according to the present invention through specific examples.

More specifically, in one embodiment of the present invention, based on the results confirmed in the previous study, it was investigated whether cytosine deamination activity appeared even in ABE mutants of an improved version than ABE7.10. As a result, all tested ABE mutants In addition to the adenine-correcting activity, it was confirmed that cytosine correction was induced (see Example 2).

In another embodiment of the present invention, the sequence and tertiary structural analysis of the homologous protein of TadA to investigate mutations that may affect the removal of cytosine-correcting activity through the distinction between adenine and cytosine in adenosine deaminase Based on this, variants in which various candidate mutations were introduced into TadA7.10 of ABEmax or ABEmax-m, respectively, were prepared and the adenine and cytosine correction efficiency at the target site in HEK293T cells was analyzed. As a result, it was found that, contrary to expectations, the P48R mutation in TadA7.10 of ABEmax-m substantially reduced the adenine correction efficiency while increasing the cytosine correction efficiency, resulting in high specificity for cytosine correction (see Example 3). ).

In another embodiment of the present invention, the conventionally known cytosine base correction gene scissors (CBE and CBE(ΔUGI)), adenine base correction gene scissors (ABEmax, ABEmax-UGI) and P48R mutations of the present invention produced by introducing As a result of analyzing the bases substituted in the target nucleotide sequence of the endogenous CSRNP3 gene by transfecting the cells with a total of six modified adenine base editing scissors (ABE-P48R, ABE-P48R-UGI), the improved version according to the present invention In the case of adenine base correction gene scissors (ABE-P48R, ABE-P48R-UGI), it was confirmed that the cytosine located at the 6th position from the 5' end was replaced with high efficiency, mainly immediately after thymine, not adenine (Example 4) Reference).

In another embodiment of the present invention, in order to verify the potential of ABE-P48R and ABE-P48R-UGI for the treatment of genetic diseases, a cell line mimicking a missense mutation of the TUBB6 gene in which the TT sequence is mutated to TC, respectively, and Cytosine correction efficiency after transfection of ABE-P48R-UGI and CBE (AncBE4max), or ABE-P48R and CBE (AncBE4max) of the present invention into a cell line mimicking the missense mutation of the TPO gene in which the TG sequence is mutated to TC was measured. As a result, the sophisticated cytosine correction function of ABE-P48R-UGI and ABE-P48R was confirmed compared to CBE, respectively, and the therapeutic potential for genetic diseases was confirmed through this (see Example 5).

Therefore, the above results prove the very sophisticated cytosine base editing effect of the adenine base editing gene scissors according to the present invention and its use.

Accordingly, as another aspect of the present invention, the present invention provides the adenine base correction gene scissors; and

It provides a composition for thymine-cytosine (T-C) sequence-specific cytosine base correction, including a guide RNA (single guide RNA; sgRNA).

The composition for cytosine base correction of the present invention may be to substitute thymine (T) or guanine (G) for cytosine (C) positioned immediately after thymine (T) on the target sequence depending on the presence or absence of UGI, more specifically As such, when UGI is present, the cytosine at the above position may be substituted with thymine, and when UGI is not present, the cytosine at the above position may be substituted with guanine.

In the present invention, the cytosine may be cytosine (C) located at the 5th to 7th positions from the 5' end of the target sequence, preferably the cytosines located at the 5th and 6th positions, more preferably It may be a cytosine located in the 6th position, but is not limited thereto.

The “guide RNA (gRNA)” is a single-stranded RNA that guides the Cas protein to the target DNA by finding the location of a specific DNA to be edited, and the guide RNA is a protospacer adjacent motif (PAM). ) adjacent to the site, and may include a sequence complementary to the nucleotide sequence of 10 to 25 bp of the DNA to be edited.

In addition, the present invention provides a thymine-cytosine (TC) sequence-specific cytosine base correction method comprising the step of contacting the composition with a target sequence in vitro .

In the base correction method according to the present invention, the target sequence may include a target base to be corrected, and the target base to be corrected is a base other than cytosine associated with a disease or disease, preferably thymine or guanine. It may be a cytosine-point mutated base, but is not limited thereto.

In addition, the present invention, comprising the composition, thymine-cytosine (T-C) provides a kit for sequence-specific cytosine base correction.

In the present invention, the kit may include all materials (reagents) necessary for performing base correction such as a buffer and deoxyribonucleotide-5-triphosphate (dNTP) together with the base correction composition. . In addition, the optimal amount of reagents to be used in a particular reaction of the kit can be readily determined by a person skilled in the art having the teachings herein.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Experimental materials and methods

1-1. Construction of plasmids for expression of ABE variants

We found that pCMV_ABEmax_P2A_GFP (Addgene # 112101), pCMV-ABEmax (TadA E59A) (Addgene #125648), pCMV-ABEmax (TadA, eTadAE59A) (Addgene #125662), pCMV-ABEmax (TadA E59A, eTadAR 47Q) (Addgene #125657 E59A, eTadAR 47Q) ), pCMV-ABEmax (TadA E59A, eTadAD108Q) (Addgene #125655), pCMV-ABEmaxAW (Addgene #125647), ABE8e (Addgene #138489), ABE8e (TadA-8e V106W) (Addgene #138495), or ABE8.17 ABE variant expression plasmid was constructed based on -m (Addgene #136298). To obtain the vector for ABE expression, 1 μg of pCMV_ABEmax_P2A_GFP, 1 unit of NotI-HF (New England Biolabs, catalog number: R3189L) and 1 unit of BglII (Enzynomics, catalog number: R010S) were mixed in a final volume of 50 μl. Then, the mixture was incubated at 37° C. for 1 hour to induce a cleavage reaction, and then electrophoresis was performed to separate the cleaved product according to size, and Expin ^TM Gel SV mini, a gel extraction kit (GeneAll, catalog number: 102- 102) was used for purification. 2U T5 exonuclease (New England Biolabs, catalog number: M0363S), 12.5U Phusion DNA polymerase (Polymerase) (Thermo Fisher Scientific, catalog number: F530L), 2kU Taq DNA ligase (New England Biolabs, catalog number: M0208S), 0.2 M Tris-HCl (pH 7.5), 0.2 M MgCl ₂ , 2 mM dNTPs, 0.2 M dithiothreitol, 25% PEG-8000, and 1 mM NAD were mixed to a final 10 μl volume and the mixture was The purified product and the synthesized PCR product containing the variant to be confirmed were mixed, and incubated at 50° C. for 1 hour. The product was then transformed into 100 μl of DH5α competent cells. Transformed single colonies were inoculated into LB medium containing antibiotics, and plasmids were isolated from the cells using a DNA prep kit (Enzynomics, EP101-200N).

1-2. Construction of ABE-UGI through DNA cloning using CRISPR-Cas9

Restriction enzyme-free DNA cloning using CRISPR-Cas9 was performed with some modifications of previously known methods. More specifically, the sgRNA was designed to cleave the sequence encoding the C-terminus of Cas9 (D10A) in pCMV_ABEmax_P2A_GFP, and 0.7 μg of in vitro transcribed sgRNA and 1 μg of recombinant NG for in vitro DNA cleavage. -SpCas9 was pre-incubated at room temperature for 5 minutes, then 1 μg of pCMV_ABEmax_P2A_GFP and DEPC-treated water were added to the SpCas9-sgRNA, and the final volume was adjusted to 50 μl. Thereafter, the mixture was incubated at 37° C. for 30 minutes to induce cleavage, and the product was purified through electrophoresis, and the inserted PCR product was amplified from pCMV_AncBE4max (Addgene plasmid # 112094). The two fragments were linked via Gibson assembly.

1-3. Transfection using liposomes

HEK293T (ATCC®CRL-3216 ^TM ) cells were cultured in DMEM medium supplemented with 10% FBS and 1% ampicillin at 37° C. and 5% CO ₂ conditions. Cell density was estimated through a hemocytometer and microscopic observation. Before transfection, HEK293T cells were seeded in 24-well plates at a density of 1×10 ⁵ cells per well and incubated for 24 hours. and 250 ng of sgRNA expression plasmid) and a mixture of serum-free medium were treated with the cells. Next, genomic DNA was isolated 72 hours after transfection.

1-4. Transfection via electroporation

The ABEmax expression plasmid (500 ng) and sgRNA expression plasmid (170 ng) were introduced into 2×10 ⁵ cells using the Neon ^TM Transfection System 10 μL kit (Thermo Fisher Scientific, MPK1025) by electroporation. Appropriate electroporation parameters (1,500V-20ms-2pulses for HEK293T cells) were performed according to the manufacturer's protocol. In addition, genomic DNA was isolated 72 hours after transfection.

1-5. Sequence alignment of TadA homologous proteins

TadA homologous proteins (orthologs) were searched using Protein BLAST of the National Center for Biotechnology Information (NCBI). E. coli wtTadA sequence (GenBank ID: WP_001297409.1) was used as the input sequence and 10 homologous proteins exhibiting ~40% sequence identity were selected. The accession numbers and species for the selected sequences are as follows: vsTadA, WP_127165941.1, Veillonella sp. CHU732; ssTadA, WP_105128341.1, Streptococcus suis ; asTadA, WP_067866801.1, Acinetobacter sp. SFB; bfTadA, WP_073388705.1, Butyrivibrio fibrisolvens ; sxTadA, WP_107541930.1, Staphylococcus xylosus ; cbTadA, WP_111988333.1, Cellvibrionaceae bacterium AOL6; dfTadA, WP_117494436.1, Dorea formicigenerans ; ocTadA, WP_047980683.1, Ornithinibacillus contaminans ; osTadA, WP_077602817.1, Oceanobacillus sojae ; pgTadA, WP_026908502.1, Paucisalibacillus globulus .

1-6. Targeted deep sequencing

HEK293T cells were centrifuged, and the cell pellet was resuspended in 100 μl of Proteinase K extraction buffer [40 mM Tris-HCl (pH 8.0), 1% Tween-20, 0.2 μM EDTA, 10 mg Proteinase K, 0.2% Nonidet P-40]. Next, incubated at 60° C. for 15 minutes and then incubated at 98° C. for 5 minutes. Genomic DNA isolated from HEK293T cells was amplified using KOD-Multi & Epi (TOYOBO, KME-101), and the resulting PCR product was analyzed using Illumina Mini-Seq. Mini-seq results were analyzed using BE-Analyzer (http://www.rgenome.net/be-analyzer/).

1-7. Targeted RNA sequencing

In a 24-well plate, HEK293T cells were transfected with 500 ng of ABE expression plasmid and 170 ng of sgRNA by electroporation, and cells were washed with DPBS after 24 hours. RNA was isolated using the NucleoSpin® RNA Plus kit (MACHEREY-NAGEL, 740984. 250) according to the manufacturer's instructions, and reverse transcription was performed using ReverTra Ace-α- ^TM (TOYOBO, FSK-101) according to the manufacturer's instructions. cDNA synthesis was performed. For cDNA amplification, PCR was performed with KOD-Multi & Epi (TOYOBO, KME-101), and the PCR product was analyzed using an Illumina Mini-Seq instrument. To obtain the ratio of adenosine to inosine edited, the number of adenosine to guanosine conversions in the product was divided by the total number of adenosine.

Example 2. Measurement of cytosine deamination activity against various ABE variants

In a previous study, the present inventors found that ABE7.10, which is adenine base editors (ABE), has a narrow working range of the preferred motif (TC * N) (5' end of the Cas9 target range as shown in FIG. 1 ) 5-7), in addition to correcting the original target base, adenine, it was confirmed that cytosine can be substituted by catalyzing cytosine deamination when there is a TC target motif. In addition, it was confirmed that cytosine base substitution by this cytosine deamination catalyst appeared not only in ABE7.10, but also in its previous versions (ABE6.3, ABE7.8 and ABE7.9) and more optimized versions (ABEmax). .

Accordingly, the present inventors have recently attempted to investigate whether cytosine deamination activity appears in ABE mutants developed by variously improving TadA, an adenosine deamination enzyme, for various purposes. The types of ABE variants used in this experiment are as follows, and detailed TadA information contained in each variant is shown in FIG. 2: 1) A version developed for the purpose of reducing the ABE-mediated RNA off-target effect (i.e., ABEmax -F148A, ABEmax-AW, and SECURE-ABEs), 2) a version comprising a TadA8e variant exhibiting increased deamination activity (i.e., ABE8e and ABE8e-V106W), and 3) TadA8s exhibiting enhanced editing activity version (i.e., ABE8.17-m).

To this end, all ABE variants were tested in human HEK293T cells against two representative endogenous targets (FANCF and RNF2) that contained both adenine residues and cytosine target motifs within the corrective action range, and high-throughput sequencing was performed to The following results were obtained.

First, as can be seen in Figure 2, all of the tested ABE variants were still shown to induce cytosine correction in addition to adenine correction, but in the case of ABEmax-F148A and ABEmax-AW, the cytosine correction efficiency was slightly reduced. confirmed that. These results were judged to support our hypothesis that further engineering of eTadA could eliminate or minimize cytosine deamination activity.

Second, all ABE8 variants showed significantly increased adenine-correcting effect compared to the previous version, but also increased cytosine-correction efficiency. Nevertheless, in the case of ABE8e-V106W, it was confirmed that the cytosine correction efficiency was reduced compared to that of ABE8e.

Third, it was found that inactivation or removal of wtTadA did not interfere with DNA editing activity, but rather, the ABEmax version without wtTadA (ABEmax-m) showed higher DNA editing activity than the original ABEmax.

Example 3. Identification of mutations in TadA that have a significant effect on cytosine correction efficiency

The present inventors tried to identify a major mutation that promotes the distinction between adenine and cytosine in TadA, an adenosine deamination enzyme, and thought that some of the TadA homologous proteins may have already evolved to avoid cytosine correction. Therefore, for this purpose, the amino acid sequences of TadA homologous proteins derived from various species were investigated.

As a result, as can be seen in FIG. 3A , based on the aligned amino acid sequence of each TadA homologous protein and the tertiary structure of Staphylococcus aureus TadA (saTadA) bound to the tRNA fragment of FIG. 3B, it is substantially active between homologous proteins. It was found that several residues inside and outside the site were variously mutated. For example, in E. coli wtTadA, P48 is substituted with arginine in most of the TadA homologous proteins, and D108 is substituted with asparagine, glutamate or serine in other homologous proteins. Could know. In addition, the saTadA structure in Fig. 3b provided insight into the structural change of the RNA substrate required for deamination of cytosine smaller than adenine. For adenine deamination activity, the hexagonal ring of adenine must be located deep inside the adenine binding pocket, similar to that seen in the saTadA structure with a purine base attached to the pocket. However, for cytosine deamination, the pyrimidine ring must be in the same position as the hexagonal ring of the purine base in the structure, resulting in a shift of the sugar-phosphate backbone to the pocket edge. We therefore decided to replace P48 and D108 with larger residues that could prevent the DNA backbone from approaching the pocket rim. In addition, V30 and F84 located in the adenine binding pocket were substituted with isoleucine and leucine found at corresponding positions in many TadA homologous proteins. Moreover, mutations previously tested and shown to incompletely reduce RNA editing activity, such as the R47Q mutation, which retains DNA on-target editing activity, and the D53E mutation, which reduces RNA editing activity in vitro, were co-introduced.

Next, each of the above candidate mutations likely to affect the cytosine-correcting effect of ABE was introduced into TadA7.10 of ABEmax or ABEmax-m, and then transfected into HEK293T cells, and each ABE variant at the target site in the FANCF and RNF2 genes. was tested for the nucleotide conversion activity of

As a result, as shown in FIG. 4, when the adenine and cytosine correction efficiency of the ABE mutant was corrected to the efficiency of ABEmax, it was confirmed that the adenine and cytosine correction efficiency was simultaneously increased or decreased in most mutants. . However, the present inventors unexpectedly found that the P48R mutation in TadA7.10 of ABEmax-m substantially reduced the adenine correction efficiency while increasing the cytosine correction efficiency, resulting in high specificity for cytosine correction.

Through the above results, TadA7.10-P48R, a mutant TadA7.10 showing improved selectivity for cytosine correction, was found.

Example 4. Confirmation of TC-specific cytosine correction effect of adenine base correction gene scissors introduced with P48R mutation

The present inventors, based on the results of Example 3, using a TadA7.10-P48R mutant introduced with a P48R mutation significantly reduced adenine-correcting activity while increasing cytosine-correcting activity TC sequence-specific base correction tool wanted to develop. For this, two copies of uracil DNA glycosylase (UGI) were ligated to the C-terminus of ABEmax-P48R, such as AncBE4max, which is an optimized cytosine base gene scissors (CBE). The addition of UGI may increase the efficiency of cytosine (C) to thymine (T) calibration rather than cytosine (C) to guanine (G) calibration, and vice versa. Therefore, the present inventors ultimately prepared the following six types as TC-specific nucleotide correction gene scissors, and schematically shown their components in FIG. 5 : CBEs AncBE4max and AncBE4max (ΔUGI), ABEs types ABEmax and ABEmax-UGI, and ABEs into which P48R was introduced, ABEmax-P48R and ABEmax-P48R-UGI.

After transfecting the six types of TC-specific nucleotide correction gene scissors into HEK293T cells, a test for nucleotide correction at the target site of the CSRNP3 gene was performed, followed by high-throughput sequencing.

As a result, as can be seen in the heat map result of FIG. 6, CBE and CBE (ΔUGI) substituted all cytosines (C) (eg, C3, C6 and C7) at the highest ratio within the corrective operating range, It was shown that ABEmax and ABEmax-UGI replaced all As (eg A4 and A8) and C6. On the other hand, it was confirmed that ABE-P48R and ABE-P48R-UGI mainly substituted C6.

Accordingly, the present inventors performed a test by adding target sites of three endogenous genes (FANCF, RNF2 and ABLIM3) to the CSRNP3 gene, and performed a high-throughput sequencing analysis. As a result, as shown in Fig. 7, the trend of corrective activity coincided with the results at the target site of the CSRNP3 gene, and in the case of ABE-P48R, mainly C was substituted with G, whereas in the case of ABE-P48R-UGI, C to T as expected. It was found that substituted with, and the same cytosine substitution effect as shown in FIG. 8, when treated with a target having a TC motif at a different position, 5' to 7th from the 5' end It can also be applied to cytosine, and as shown in FIG. 9 , it was confirmed that a target-specific cytosine correction effect having a TC motif occurred.

These results suggest that ABE-P48R and ABE-P48R-UGI could serve as correction tools for substitution of guanine and thymine for cytosine directly adjacent to thymine (TC-to-TG and TC-to-TT), respectively. suggesting that there is

Example 5. Validation of the possibility of treating genetic diseases using adenine base editing gene scissors with enhanced TC-specific cytosine base editing activity

To validate the potential of ABE-P48R and ABE-P48R-UGI for the treatment of genetic diseases, we investigated all targeting variants registered in the in silico ClinVar database. As can be seen in FIG. 10 , a total of 36,153 T > C mutations that caused the pathological phenotype in the database were located in the standard cytosine base correction operating range (4th to 8th). Of these, 3,874 (11%) mutations were associated with cytosine target motifs within the ABE-P48R-UGI correction operating range. It was also confirmed that 3,248 (14%) of 23,237 G > C mutations in the database could be targeted by ABE-P48R.

5-1. Calibration from TC to TT using ABE-P48R-UGI

For example, a missense mutation in the TUBB6 gene (causing a F394S change in the protein) is associated with congenital facial paralysis, bilateral ptosis, and palatopharyngeal dysfunction. For radical treatment, the TC sequence must be corrected with TT. Accordingly, the present inventors first established a cell line containing an appropriate mutation in the genome to mimic the genetic variation of the disease, and then transfected the cell line with CBE (AncBE4max) or ABE-P48R-UGI, respectively, and then, high-throughput A nucleotide sequence analysis was performed.

As a result, as shown in FIG. 11 , it was confirmed that CBE showed a higher total cytosine correction efficiency than ABE-P48R-UGI, but the correct correction efficiency was lower (ABE-P48R-UGI was 4.1%, CBE is less than 1%). In addition, it was confirmed that ABE-P48R-UGI showed a negligible bystander effect, whereas CBE showed a lot of bystander effect. Therefore, the above results suggest that ABE-P48R-UGI can be used as a useful calibration tool for TC to TT calibration.

5-2. Calibration from TC to TG with ABE-P48R

For missense mutations in the TPO gene (causing the Q660E change) found in patients with both nontoxic and toxic goiter, the TC sequence must be corrected with TG for treatment. Accordingly, the present inventors established a cell line containing an appropriate mutation in the genome to mimic the genetic variation of the disease as described above, and then transfected the cell line with CBE (AncBE4max) or ABE-P48R, respectively, and then Throughput sequencing was performed.

As a result, as can be seen in FIG. 12 , compared to ABE-P48R, CBE showed higher total cytosine correction efficiency, but confirmed that the correct correction rate was lower (3.1% for ABE-P48R, 1% for CBE) under). In addition, ABE-P48R showed a negligible bystander effect like ABE-P48R-UGI, whereas CBE showed a lot of bystander effect. suggesting that there is

Claims (9)

adenosine deaminase variants comprising a P48R mutation; and
Cas9 (CRISPR associated protein 9) protein fused, thymine-cytosine (TC) sequence-specific cytosine editing activity enhanced adenine base editing (Adenine Base Editor).
According to claim 1,
The adenosine deamination enzyme is TadA7.10, characterized in that, adenine base correction gene scissors.
According to claim 1,
The gene scissors are adenine base correction gene scissors, characterized in that the mutation is introduced in ABEmax.
According to claim 1,
The gene scissors is characterized in that one or more uracil DNA glycosylase (uracil DNA glycosylase; UGI) is further linked, adenine base correction gene scissors.
The adenine base correction gene of any one of claims 1 to 4; and
A composition for thymine-cytosine (TC) sequence-specific cytosine base correction comprising a guide RNA (single guide RNA; sgRNA).
6. The method of claim 5,
The cytosine is characterized in that the cytosine (C) located at the 5th to 7th position from the 5' end of the target sequence, the composition.
6. The method of claim 5,
The composition is characterized in that the cytosine (C) positioned immediately after thymine (T) on the target sequence is substituted with thymine (T) or guanine (G) depending on the presence or absence of UGI.
A thymine-cytosine (TC) sequence-specific cytosine base correction method comprising the step of contacting the composition of claim 5 with a target sequence in vitro .
Comprising the composition of claim 5, thymine-cytosine (T-C) sequence-specific cytosine base correction kit.

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