WO2023036189A1 - Adenine deaminase, adenine base editor containing same, and applications thereof - Google Patents

Adenine deaminase, adenine base editor containing same, and applications thereof Download PDF

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WO2023036189A1
WO2023036189A1 PCT/CN2022/117592 CN2022117592W WO2023036189A1 WO 2023036189 A1 WO2023036189 A1 WO 2023036189A1 CN 2022117592 W CN2022117592 W CN 2022117592W WO 2023036189 A1 WO2023036189 A1 WO 2023036189A1
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adenine
amino acid
editing
deaminase
base
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陈亮
李大力
张舜
薛念念
陈曦
刘明耀
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华东师范大学
上海邦耀生物科技有限公司
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Definitions

  • 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.
  • 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.
  • 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.
  • 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, including the adenine base Editors and their applications.
  • 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 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, described adenine deaminase comprises the 29th, the 84th, the 108th and the 145th of the amino acid sequence shown in SEQ ID NO:1 There are one or more amino acid differences.
  • the amino acid difference includes that the 108th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is Q, H, S, A, C, D, E, F, G , I, K, L, M, P, R, S, T, V, W or Y.
  • the amino acid difference includes that the 145th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is T, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, V, W, or Y.
  • the amino acid difference includes that the 84th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is T.
  • the multiple amino acid differences include that the 84th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is T, and the 108th amino acid residue is Q.
  • the multiple amino acid differences include that the 108th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is Q, and the 145th amino acid residue is T.
  • the multiple amino acid differences include that the 108th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is Q, and the 29th amino acid residue is M.
  • the multiple amino acid differences include that the 108th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is Q, and the 29th amino acid residue is W.
  • amino acid sequence is as shown in SEQ ID NO:1 and the nucleotide sequence of adenine deaminase is as shown in SEQ ID NO:2.
  • the adenine deaminase also includes the presence of one or more amino acids at positions other than the 29th, 84th, 108th and 145th as shown in SEQ ID NO:1 Mutation difference, the formed mutant has the same or similar function or biological activity as the adenine deaminase described in the first aspect.
  • 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.
  • the nuclease is a Cas protein or a variant thereof.
  • 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;
  • the protein variant is VQR-spCas9, VRER-spCas9, spRY, spNG, SaCas9-KKH or SaCas9-NG.
  • the adenine base editor significantly reduces bystander adenine editing and bystander cytosine editing.
  • the adenine base editor can greatly narrow the editing range, precisely edit 1-2 bases, and keep the occurrence of indel events low.
  • the adenine base editor can expand the editing range and improve the editing efficiency.
  • the third aspect of the present invention provides a fusion protein comprising the adenine deaminase as described in the first aspect.
  • the fusion protein further comprises a nuclease, and the nuclease is as described in the second aspect.
  • the promoter can be conventional in the field, such as CMV or other types of spectral promoters and tissue-specific promoters, such as CAG, PGK, EF1 ⁇ ; muscle-specific promoter Ctsk; liver-specific promoter Lp1, etc. .
  • the polyA can be conventional in the field, such as bovine growth hormone polyadenylation signal BGH polyA, or polyadenylation signal of other biological sources.
  • the sequence consists of CMV-adenine deaminase-Cas9n-BGH polyA.
  • a fourth aspect of the present invention provides an adenine base editing system, comprising: sgRNA and the adenine base editor as described in the second aspect.
  • the target sequence of the sgRNA is shown in any one 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 base editing method, the base editing method comprising:
  • the purine base editing system enables base editing of the target cells.
  • the base editing method further includes adding sgRNA, and the target sequence of the sgRNA is shown in any one of SEQ ID NO: 4-15.
  • the source of the target cells is an isolated cell line.
  • the base editing method is a non-therapeutic base editing method.
  • 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.
  • the base editing method can also be used for therapeutic purposes.
  • 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.
  • the target cells may be eukaryotic cells, prokaryotic cells, or archaic cells different from prokaryotic cells.
  • the target cell can express the fusion protein as described in the third aspect.
  • the target cells may be plant cells, human cells or animal cells.
  • the seventh aspect of the present invention provides an 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 eighth aspect of the present invention provides an 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 ninth aspect of the present invention provides an 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 Application of the adenine base editing system in the preparation of base editing tools.
  • the reagents and raw materials used in the present invention are all commercially available.
  • 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, broaden the editing range, and maintain a high Editing activity and editing precision; the structural pocket changes caused by mutations also make adenine deaminase unable to recognize cytosine as a substrate, and finally completely eliminate the independent cytosine editing events in ABE; and maintain a low indel, improve Safety can promote its application in precision medicine, animal disease model making, crop genetics and breeding, etc., and has great application value.
  • 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.
  • Figure 9 is a schematic diagram of the comparison results of A>G editing efficiency achieved by the ABE variants on the ABE-site3 endogenous target on 293T.
  • the identification primers of the targets used in the examples are shown in Table 3.
  • F is the forward primer
  • R is the reverse primer
  • the seamless cloning kit used in the examples is Vazyme ClonExpress MultiS One Step Cloning Kit, C113-01.
  • the HEK293T cells used in the examples are ATCC CRL-3216 cell lines.
  • the nucleotide sequence of the plasmid U6-sgRNA-EF1 ⁇ -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.
  • the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Afterwards, 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 products, and then use the round of PCR products as templates , 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.
  • Hi-Tom Gene Editing Detection Kit Novogene
  • ABE8e-N108Q was selected to be verified again in four other endogenous targets (EGFR-library-sg4, HBG1-sg1, EMX1-sg2p, HBG-sg8).
  • 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%.
  • ABE8e-L145T, ABE8e-L145Q, ABE8e-N108Q and ABE8e-F84T all significantly reduced bystander adenine editing and bystander cytosine editing.
  • This embodiment is designed to completely eliminate bystander cytosine editing and to achieve precise editing of a single A>G.
  • HEK293T cells were digested and inoculated into 96-well plates at 2 ⁇ 10 5 cells/well.
  • the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Afterwards, use the operation process of Hitom kit to design corresponding identification primers (as shown in table 3), that is, add the bridge sequence shown in SEQ ID NO:38 at the 5' end of the forward primer, and the reverse primer 5' Add the bridging sequence shown in SEQ ID NO: 39 to the end to obtain a round of PCR product, 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, and then mix them together for gel cutting, recovery and purification Sent to the company for sequencing.
  • DP304 Tiangen Cell Genome Extraction Kit
  • 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 .
  • 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
  • the four combined mutations only edited 1 base, and edited A5 efficiently and accurately.
  • ABE8e covers ⁇ 7 bases
  • ABE8e-F148A targets ⁇ 4 bases
  • the editing range is still ⁇ 3 bases
  • N108Q-L145T targets ⁇ 4 bases
  • 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.
  • ABE8e-N108Q was named ABE9s
  • 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.
  • 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.
  • This embodiment is designed to compare the working characteristics of ABE9.1s, ABE9.2s, ABE9.3s and ABE9.4s.
  • HEK293T cells were digested and inoculated into 96-well plates at 2 ⁇ 10 5 cells/well.
  • the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Afterwards, use the operation process of Hitom kit to design corresponding identification primers (as shown in table 3), that is, add the bridge sequence shown in SEQ ID NO:38 at the 5' end of the forward primer, and the reverse primer 5' Add the bridging sequence shown in SEQ ID NO: 39 to the end to obtain a round of PCR product, 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, and then mix them together for gel cutting, recovery and purification Then send it to the company for sequencing.
  • Hitom kit to design corresponding identification primers (as shown in table 3), that is, add the bridge sequence shown in SEQ ID NO:38 at the 5' end of the forward primer, and the reverse primer 5' Add the bridging sequence shown in SEQ ID NO: 39 to the end to obtain a round of PCR product, then use the round of PCR product as a template
  • 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
  • the editing range of ABE8e is ⁇ 5 bases
  • the coverage of ABE9s is 3 to 4 bases
  • ABE9.1s, ABE9.2s, ABE9.3s and In ABE9.4s except ABE9.1s with slight A7 or A4 editing, the other three variants only edited one base.
  • 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.
  • 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 ).
  • the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Afterwards, use the operation process of Hitom kit to design corresponding identification primers (as shown in table 3), that is, add the bridge sequence shown in SEQ ID NO:38 at the 5' end of the forward primer, and the reverse primer 5' Add the bridging sequence shown in SEQ ID NO: 39 to the end to obtain a round of PCR product, 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, and then mix them together for gel cutting, recovery and purification Then send it to the company for sequencing.
  • Hitom kit to design corresponding identification primers (as shown in table 3), that is, add the bridge sequence shown in SEQ ID NO:38 at the 5' end of the forward primer, and the reverse primer 5' Add the bridging sequence shown in SEQ ID NO: 39 to the end to obtain a round of PCR product, then use the round of PCR product as a template
  • the corresponding ABE8e editing activity window is A4-A7 bases
  • ABE9.1 still mainly edits A5 precisely, and almost all N108 site mutations compared with ABE8e All can effectively narrow the editing range, and some variants maintain high efficiency (such as N108H, N108Q, N108S, etc.), and among L145 saturation mutations, mutations such as L145D, L145E, and L145G also narrow the editing range compared with ABE8e.
  • L145W significantly expanded the active window (A4-A9), and the editing efficiency of A8 and A9 was greatly improved, which were 4.51 times and 6.03 times higher than ABE8e, respectively.
  • N108 and L145 mutations play a crucial role in regulating ABE editing properties (window and activity, etc.), such as most N108 mutants and L145D, L145E, L145G and other L145 mutants, It can narrow the editing window and improve the editing accuracy, while L145W can further expand the editing range and greatly improve the editing efficiency, which enables efficient targeting of adenine that was previously uneditable near the PAM region and expands the application range of ABE.

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Abstract

Provided are adenine deaminase, an adenine base editor containing same, and applications thereof. Adenine deaminase has one or more amino acid differences at positions 29, 84, 108 and 145, which comprise the amino acid sequence as shown in SEQ ID NO:1. The adenine base editor comprises a nuclease and adenine deaminase, and can effectively undermine the non-specific binding of adenine deaminase to substrate bases, and narrow an editing window to 1-2 bases while maintaining high editing activity. Structural pocket changes caused by mutations also prevent adenine deaminase from recognizing cytosine as a substrate, and finally completely eliminate independent cytosine editing events present in ABE. Moreover, indel is kept low, safety is improved, and the applications in precise medical treatments, animal disease model making, crop genetic breeding, and the like can be promoted, which has great application value.

Description

腺嘌呤脱氨酶、包含其的腺嘌呤碱基编辑器及其应用Adenine deaminase, adenine base editor comprising it and application thereof
本申请要求申请日为2021/9/7的中国专利申请2021110442060的优先权。本申请引用上述中国专利申请的全文。This application claims the priority of the Chinese patent application 2021110442060 with the filing date of 2021/9/7. This application cites the full text of the above-mentioned Chinese patent application.
技术领域technical field
本发明属于基因编辑领域，具体涉及一种腺嘌呤脱氨酶、包含其的腺嘌呤碱基编辑器及其应用。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.
背景技术Background technique
人类遗传病发生的本质是由于基因突变，60％左右的遗传疾病由单个碱基突变引起，传统的利用基因组编辑技术介导的同源重组进行纠正这类遗传病非常低效(0.1％-5％)。基于CRISPR系统衍生出来的单碱基编辑器(Base editor)是近年来新兴的高效碱基编辑技术，因其不产生DNA双链断裂、无须重组模板、高效编辑等优势，在基础研究和临床疾病治疗展示了巨大的应用前景。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.
经典的碱基编辑器主要分为胞嘧啶碱基编辑器和腺嘌呤碱基编辑器，前者由活性受损的来源于酿脓链球菌(Streptococcus pyogenes)的Cas9蛋白spCas9n、大鼠来源的胞嘧啶脱氨酶rAPOBEC1和尿嘧啶糖苷酶抑制剂组成，其中Cas9蛋白以NGG作为PAM识别并特异结合DNA，紧接着在脱氨酶以及DNA修复的作用下，最终在NGG(21～23位)上游靶向序列20bp范围实现C/G-T/A的替换，编辑窗口主要位于4～8位；后者则是将细菌来源的TadA(腺嘌呤脱氨酶)与spCas9融合，在定向进化和蛋白质工程化改造技术的辅助下，经历7轮进化最终获得可作用于单链DNA的腺嘌呤碱基编辑器ABE7.10，活性编辑区域主要位于4～7位，该系统在人类细胞中引起A/T-G/C的平均编辑效率约为53％，远高于利用同源重组介导碱基突变的效率，其产物纯度高达99.9％以及极低的indel(insertion-deletion，插入或缺失)发生，更重要的是人类致病性点突变约47％是由C·G突变为T·A所形成，而腺嘌呤碱基编辑器有望修正近一半的病原性点突变，展现出其在突变碱基修改以及遗传病治疗的巨大潜力，目前ABE已广泛应用于动物模型制备和基因治疗。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.
针对ABE7.10编辑效率低下以及编辑窗口狭小等问题，各实验室开展了大量的优化改造工作，利用更换核定位信号和密码子优化的策略获得ABEmax，相比于ABE7.10， A到G的编辑效率最高改善了7.1倍，融合CP-Cas9变体构建的CP-ABEmax系列将编辑窗口由4～7位扩展至4～12位，但编辑活性仍与ABEmax类似，此外为进一步扩大ABE的靶向范围，具备不同PAM选择性的ABE也被开发出来，如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)。借助分子进化技术，最新报道的ABE8e(Richter MF,et al.Phage-assisted evolution of an adenine base editor with improved cas domain compatibility and activity.Nat Biotechnol,2020,38:883-891)和ABE8s(Gaudelli NM,et al.Directed evolution of adenine base editors with increased activity and therapeutic application.Nat Biotechnol,2020,38:892-900)，再次显著性提高了碱基编辑效率，其中ABE8e活性相较于ABE7.10提高了590倍，同时编辑范围也进一步扩大，范围可覆盖3～14位，这也不可避免产生严重的“旁观者效应”，即窗口内所有腺嘌呤均会产生编辑，ABE8e和ABE8s执行编辑功能时均无法区别目标A和非目标A，因此对于精准医疗的临床应用仍缺少高精度高活性的腺嘌呤碱基编辑器，与此同时多个课题组报道ABE存在危害性的胞嘧啶编辑(Li S,et al.Docking sites inside cas9 for adenine base editing diversification and rna off-target elimination.Nat Commun,2020,11:5827；Kurt IC,et al.Crispr c-to-g base editors for inducing targeted DNA transversions 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)，这也势必引发ABE应用安全性的担忧。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 domain compatibility and activity. Nat Biotechnol, 2020, 38:883-891) and ABE8s (Gaudelli NM, 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% compared with ABE7.10 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 neither ABE8e nor ABE8s can perform editing functions. Differentiate between target A 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, multiple research groups have reported that ABE has harmful cytosine editing (Li S, et al al.Docking sites inside cas9 for adenine base editing diversification and rna off-target elimination.Nat Commun,2020,11:5827; Kurt IC,et al.Crispr c-to-g base editors for inducing targeted DNA transformations 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; Grun ewald J, et al. Crispr DNA base editors with reduced rna off-target and self-editing activities. Nat Biotechnol, 2019, 37:1041-1048), which will inevitably lead to concerns about the safety of ABE applications.
目前，尚无可缩窄ABE的编辑窗口至1～2个碱基的报道，实现精准的腺嘌呤编辑仍然缺乏有效的碱基编辑器，同时ABE产生的危害性胞嘧啶编辑也没有得到完全地消除，产生的ABE安全性问题亟待解决。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.
发明内容Contents of the invention
本发明所要解决的技术问题是为了克服现有技术中缺少能够显著降低旁观者腺嘌呤和胞嘧啶编辑，提供一种精准高效、高安全性的腺嘌呤脱氨酶、包含其的腺嘌呤碱基编辑器及其应用。本发明的腺嘌呤碱基编辑器能够极大降低旁观者腺嘌呤、几乎完全去除旁观者胞嘧啶编辑，甚至能够将编辑窗口缩窄至1～2个碱基，具有极高的安全性。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, including the adenine base Editors and their applications. 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.
发明人通过结构生物学预测了TadA-8e中几个关键性的催化位点，在此基础上，通过氨基酸替换，获得基于TadA-8e的单点突变体，意外发现部分单点突变体极大地破坏了腺嘌呤脱氨酶与底物腺嘌呤的非特异性结合，同时维持较高的编辑活性；在单点突变体的基础上进一步通过氨基酸替换获得双点突变体，发现双点突变体可将编辑窗口缩窄至1～2个碱基，同时维持较高的编辑活性，而突变引起的结构口袋变化也使得腺嘌呤脱氨酶无法识别胞嘧啶作为底物，最终完全消除了ABE中存在的独立胞嘧啶编辑事件，此外仍然保持较低的indel。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.
本发明通过以下技术方案解决上述技术问题。The present invention solves the above-mentioned technical problems through the following technical solutions.
本发明的第一方面提供一种腺嘌呤脱氨酶，所述腺嘌呤脱氨酶在包括如SEQ ID NO:1所示的氨基酸序列的第29位、第84位、第108位和第145位存在一种或多种氨基酸差异。The first aspect of the present invention provides a kind of adenine deaminase, described adenine deaminase comprises the 29th, the 84th, the 108th and the 145th of the amino acid sequence shown in SEQ ID NO:1 There are one or more amino acid differences.
本发明一些实施方案中，所述一种氨基酸差异包括如SEQ ID NO:1所示的氨基酸序列的第108位氨基酸残基为Q、H、S、A、C、D、E、F、G、I、K、L、M、P、R、S、T、V、W或Y。In some embodiments of the present invention, the amino acid difference includes that the 108th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is Q, H, S, A, C, D, E, F, G , I, K, L, M, P, R, S, T, V, W or Y.
本发明另一些实施方案中，所述一种氨基酸差异包括如SEQ ID NO:1所示的氨基酸序列的第145位氨基酸残基为T、A、C、D、E、F、G、H、I、K、M、N、P、Q、R、S、V、W或Y。In other embodiments of the present invention, the amino acid difference includes that the 145th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is T, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, V, W, or Y.
本发明另一些实施方案中，所述一种氨基酸差异包括如SEQ ID NO:1所示的氨基酸序列的第84位氨基酸残基为T。In other embodiments of the present invention, the amino acid difference includes that the 84th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is T.
本发明一些实施方案中，所述多种氨基酸差异包括如SEQ ID NO:1所示的氨基酸序列的第84位氨基酸残基为T，并且第108位氨基酸残基为Q。In some embodiments of the present invention, the multiple amino acid differences include that the 84th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is T, and the 108th amino acid residue is Q.
本发明另一些实施方案中，所述多种氨基酸差异包括如SEQ ID NO:1所示的氨基酸序列的第108位氨基酸残基为Q，并且第145位氨基酸残基为T。In other embodiments of the present invention, the multiple amino acid differences include that the 108th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is Q, and the 145th amino acid residue is T.
本发明另一些实施方案中，所述多种氨基酸差异包括如SEQ ID NO:1所示的氨基酸序列的第108位氨基酸残基为Q，并且第29位氨基酸残基为M。In other embodiments of the present invention, the multiple amino acid differences include that the 108th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is Q, and the 29th amino acid residue is M.
本发明另一些实施方案中，所述多种氨基酸差异包括如SEQ ID NO:1所示的氨基酸序列的第108位氨基酸残基为Q，并且第29位氨基酸残基为W。In other embodiments of the present invention, the multiple amino acid differences include that the 108th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is Q, and the 29th amino acid residue is W.
本发明中，氨基酸序列如SEQ ID NO:1所示的腺嘌呤脱氨酶的核苷酸序列如SEQ ID NO:2所示。In the present invention, the amino acid sequence is as shown in SEQ ID NO:1 and the nucleotide sequence of adenine deaminase is as shown in SEQ ID NO:2.
本发明中，所述腺嘌呤脱氨酶还包括在如SEQ ID NO:1所示的除第29位、第84位、第108位和第145位的其他位点存在一种或多种氨基酸突变差异，形成的突变体具 有与第一方面所述的腺嘌呤脱氨酶相同或相近的功能或生物学活性。In the present invention, the adenine deaminase also includes the presence of one or more amino acids at positions other than the 29th, 84th, 108th and 145th as shown in SEQ ID NO:1 Mutation difference, the formed mutant has the same or similar function or biological activity as the adenine deaminase described in the first aspect.
在本发明一些实施方案中，所述腺嘌呤脱氨酶还包括核定位信号序列。In some embodiments of the present invention, the adenine deaminase further includes a nuclear localization signal sequence.
本发明中，所述核定位信号序列可为本领域常规，例如为如SEQ ID NO:3所示的核定位信号序列。In the present invention, the nuclear localization signal sequence can be conventional in the art, for example, the nuclear localization signal sequence shown in SEQ ID NO:3.
本发明的第二方面提供一种腺嘌呤碱基编辑器，所述腺嘌呤碱基编辑器包括核酸酶和如第一方面所述的腺嘌呤脱氨酶。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.
在本发明一些实施方案中，所述核酸酶为Cas蛋白或其变体。In some embodiments of the present invention, the nuclease is a Cas protein or a variant thereof.
在本发明一些较佳实施方案中，所述Cas蛋白为酿酒酵母来源的spCas9、金黄色葡萄球菌来源的SaCas9、毛螺菌科细菌来源的LbCas12a或酸胺球菌属细菌来源的enAsCas12a；所述Cas蛋白变体为VQR-spCas9、VRER-spCas9、spRY、spNG、SaCas9-KKH或SaCas9-NG。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; The protein variant is VQR-spCas9, VRER-spCas9, spRY, spNG, SaCas9-KKH or SaCas9-NG.
在本发明一些实施方案中，所述腺嘌呤碱基编辑器显著降低旁观者腺嘌呤编辑和旁观者胞嘧啶编辑。In some embodiments of the invention, the adenine base editor significantly reduces bystander adenine editing and bystander cytosine editing.
在本发明另一些实施方案中，所述腺嘌呤碱基编辑器可极大缩窄编辑范围，精准编辑1～2个碱基，同时保持较低的indel事件发生。In other 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.
本发明另一些实施方案中，所述腺嘌呤碱基编辑器可扩大编辑范围，提高编辑效率。In other embodiments of the present invention, the adenine base editor can expand the editing range and improve the editing efficiency.
本发明的第三方面提供一种融合蛋白，所述融合蛋白包含如第一方面所述的腺嘌呤脱氨酶。The third aspect of the present invention provides a fusion protein comprising the adenine deaminase as described in the first aspect.
在本发明一些实施方案中，所述融合蛋白还包含核酸酶，所述核酸酶如第二方面所述。In some embodiments of the present invention, the fusion protein further comprises a nuclease, and the nuclease is as described in the second aspect.
本发明中，编码所述融合蛋白的序列组成可以为启动子-腺嘌呤脱氨酶-核酸酶-polyA，只要其能提供不低于ABE8e的A>G的编辑效率。In the present invention, the sequence composition of 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.
本发明中，所述启动子可为本领域常规，例如CMV或者其他类型的光谱启动子及组织特异性启动子，例如CAG、PGK、EF1α；肌肉特异启动子Ctsk；肝脏特异性启动子Lp1等。In the present invention, the promoter can be conventional in the field, such as CMV or other types of spectral promoters and tissue-specific promoters, such as CAG, PGK, EF1α; muscle-specific promoter Ctsk; liver-specific promoter Lp1, etc. .
本发明中，所述polyA可为本领域常规，例如牛生长激素多腺苷酸化信号BGH polyA，或者其他生物来源的多腺苷酸化信号。In the present invention, the polyA can be conventional in the field, such as bovine growth hormone polyadenylation signal BGH polyA, or polyadenylation signal of other biological sources.
本发明一些具体实施方案中，所述序列组成为CMV-腺嘌呤脱氨酶-Cas9n-BGH polyA。In some specific embodiments of the present invention, the sequence consists of CMV-adenine deaminase-Cas9n-BGH polyA.
本发明的第四方面提供一种腺嘌呤碱基编辑系统，其包括：sgRNA和如第二方面所 述的腺嘌呤碱基编辑器。A fourth aspect of the present invention provides an adenine base editing system, comprising: sgRNA and the adenine base editor as described in the second aspect.
在本发明一些实施方案中，所述sgRNA的靶序列如SEQ ID NO:4～15任一项所示。In some embodiments of the present invention, the target sequence of the sgRNA is shown in any one of SEQ ID NO: 4-15.
本发明的第五方面提供一种药物组合物，所述药物组合物包括如第一方面所述的腺嘌呤脱氨酶、如第二方面所述的腺嘌呤碱基编辑器、如第三方面所述的融合蛋白或者如第四方面所述的腺嘌呤碱基编辑系统。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.
本发明的第六方面提供一种碱基编辑方法，所述碱基编辑方法包括：A sixth aspect of the present invention provides a base editing method, the base editing method comprising:
在靶细胞中表达如第一方面所述的腺嘌呤脱氨酶、如第二方面所述的腺嘌呤碱基编辑器、如第三方面所述的融合蛋白或者如第四方面所述的腺嘌呤碱基编辑系统，使所述靶细胞发生碱基编辑。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 enables base editing of the target cells.
本发明一些实施方案中，所述碱基编辑方法还包括加入sgRNA，所述sgRNA的靶序列如SEQ ID NO:4～15任一项所示。在本发明一些实施方案中，所述靶细胞的来源为分离的细胞系。In some embodiments of the present invention, the base editing method further includes adding sgRNA, and the target sequence of the sgRNA is shown in any one of SEQ ID NO: 4-15. In some embodiments of the invention, the source of the target cells is an isolated cell line.
在本发明一些较佳的实施方案中，所述分离的细胞系为293T细胞、HELA细胞、U2OS细胞、NIH3T3细胞或N2A细胞。In some preferred embodiments of the present invention, the isolated cell line is 293T cells, HELA cells, U2OS cells, NIH3T3 cells or N2A cells.
本发明一些实施方案中，所述碱基编辑方法为非治疗目的的碱基编辑方法。In some embodiments of the present invention, the base editing method is a non-therapeutic base editing method.
本发明中，所述非治疗目的例如在实验室中通过检测靶细胞发生的编辑来评价本发明所述的腺嘌呤脱氨酶、腺嘌呤碱基编辑器、融合蛋白或者所述的腺嘌呤碱基编辑系统。反之，也可以通过碱基编辑研究靶细胞的功能。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.
本发明中，所述碱基编辑方法还可以是治疗目的的。本发明中，所述治疗是指治疗受试者例如人类的疾病，包括抑制所述疾病的发生或发展、缓解所述疾病的症状或治愈所述疾病。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.
本发明中，所述靶细胞可以为真核细胞、原核细胞、或者不同于原核细胞的古生物细胞。In the present invention, the target cells may be eukaryotic cells, prokaryotic cells, or archaic cells different from prokaryotic cells.
较佳地，所述靶细胞可以表达如第三方面所述的融合蛋白。Preferably, the target cell can express the fusion protein as described in the third aspect.
更佳地，所述靶细胞可以为植物细胞、人类细胞或动物细胞。More preferably, the target cells may be plant cells, human cells or animal cells.
本发明的第七方面提供一种如第一方面所述的腺嘌呤脱氨酶、如第二方面所述的腺嘌呤碱基编辑器、如第三方面所述的融合蛋白或者如第四方面所述的腺嘌呤碱基编辑系统在制备碱基编辑的药物或制备基因治疗的药物中的应用。The seventh aspect of the present invention provides an 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 gene therapy drugs.
本发明的第八方面提供一种如第一方面所述的腺嘌呤脱氨酶、如第二方面所述的腺嘌呤碱基编辑器、如第三方面所述的融合蛋白或者如第四方面所述的腺嘌呤碱基编辑系 统在构建动物模型和农作物育种中的应用。The eighth aspect of the present invention provides an 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.
本发明的第九方面提供一种如第一方面所述的腺嘌呤脱氨酶、如第二方面所述的腺嘌呤碱基编辑器、如第三方面所述的融合蛋白或者如第四方面所述的腺嘌呤碱基编辑系统在制备碱基编辑工具中的应用。The ninth aspect of the present invention provides an 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 Application of the adenine base editing system in the preparation of base editing tools.
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。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.
本发明所用试剂和原料均市售可得。The reagents and raw materials used in the present invention are all commercially available.
本发明的积极进步效果在于：The positive progress effect of the present invention is:
本发明的腺嘌呤碱基编辑器可有效破坏腺嘌呤脱氨酶与底物碱基的非特异性结合，将编辑窗口缩窄至1～2个碱基，扩宽编辑范围，同时维持较高的编辑活性和编辑精度；突变引起的结构口袋变化也使得腺嘌呤脱氨酶无法识别胞嘧啶作为底物，最终完全消除了ABE中存在的独立胞嘧啶编辑事件；并且保持较低的indel，提高了安全性，可促进其在精准医疗、动物疾病模型制作、作物遗传育种等方面的应用，具有极大的应用价值。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, broaden the editing range, and maintain a high Editing activity and editing precision; the structural pocket changes caused by mutations also make adenine deaminase unable to recognize cytosine as a substrate, and finally completely eliminate the independent cytosine editing events in ABE; and maintain a low indel, improve Safety can promote its application in precision medicine, animal disease model making, crop genetics and breeding, etc., and has great application value.
附图说明Description of drawings
图1为ABE8e结合底物DNA的晶体结构(PDB:6VPC)。Figure 1 is the crystal structure of ABE8e bound to substrate DNA (PDB: 6VPC).
图2为21个ABE8e突变体在293T上FANCF site1位点实现的A>G碱基编辑对比结果示意图。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.
图3为21个ABE8e突变体在293T上FANCF site1位点实现的A4以及C6碱基编辑对比结果示意图。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.
图4为ABE8e与ABE8e-N108Q在293T上4个靶点产生的A>G、C>G、C>T、C>A碱基编辑对比结果示意图。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.
图5为19个ABE8e组合突变在293T上ABE-site3和ABE-site10位点实现的A>G碱基编辑对比结果示意图。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.
图6为ABE9s和ABE9.1s在293T上ABE-site10、HEK-site7位点实现的A碱基以及C碱基编辑效率对比结果示意图。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.
图7为ABE9s、ABE9.1s、ABE9.2s、ABE9.3s和ABE9.4s在293T上ABE-site16、ABE-site17、ABE-site13和ABE-site8内源性靶点实现的A>G编辑效率对比结果示意图。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.
图8为ABE9s、ABE9.1s、ABE9.2s、ABE9.3s和ABE9.4s在293T上ABE-site16、ABE-site17、ABE-site13和ABE-site8内源性靶点产生的indel对比结果示意图。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.
图9为ABE变体在293T上ABE-site3内源性靶点实现的A>G编辑效率对比结果示意图。Figure 9 is a schematic diagram of the comparison results of A>G editing efficiency achieved by the ABE variants on the ABE-site3 endogenous target on 293T.
具体实施方式Detailed ways
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。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.
实施例中使用的突变体中，编码突变位点的密码子如表1所示。Among the mutants used in the examples, the codons encoding the mutation sites are shown in Table 1.
表1 TadA单点和双点突变序列Table 1 TadA single-point and double-point mutation sequences
单点突变体single point mutant 密码子a 单点突变体single point mutant 密码子a 双点突变体double point mutant 密码子a
E27QE27Q cagcag L145IL145I atcatc N108Q-E27QN108Q-E27Q cag-cagcag-cag
E27RE27R agaaga L145KL145K aagaag N108Q-E27RN108Q-E27R cag-agacag-aga
V28FV28F ttcttc L145ML145M atgatg N108Q-V28FN108Q-V28F cag-ttccag-ttc
V28GV28G ggagga L145NL145N aacaac N108Q-V28GN108Q-V28G cag-ggacag-gga
V28NV28N aacaac L145PL145P cctcct N108Q-V28NN108Q-V28N cag-aaccag-aac
P29DP29D gatgat L145RL145R agaaga N108Q-P29DN108Q-P29D cag-gatcag-gat
P29MP29M atgatg L145SL145S agcagc N108Q-P29MN108Q-P29M cag-atgcag-atg
P29TP29T accacc L145VL145V gtggtg N108Q-P29TN108Q-P29T cag-acccag-acc
H57DH57D gatgat L145WL145W tggtgg N108Q-P29WN108Q-P29W cag-tggcag-tgg
H57QH57Q cagcag L145YL145Y tactac N108Q-H57DN108Q-H57D cag-gatcag-gat
F84IF84I atcatc N108AN108A gcagca N108Q-H57QN108Q-H57Q cag-cagcag-cag
F84TF84T accacc N108CN108C tgctgc N108Q-F84IN108Q-F84I cag-atccag-atc
P86GP86G ggagga N108DN108D gatgat N108Q-F84TN108Q-F84T cag-acccag-acc
N108QN108Q cagcag N108EN108E gaggag N108Q-F84VN108Q-F84V cag-gtgcag-gtg
N108TN108T accacc N108FN108F ttcttc N108Q-P86GN108Q-P86G cag-ggacag-gga
N108VN108V gtggtg N108GN108G ggagga N108Q-L145CN108Q-L145C cag-tgccag-tgc
L145CL145C tgctgc N108HN108H caccac N108Q-L145QN108Q-L145Q cag-cagcag-cag
L145QL145Q cagcag N108IN108I atcatc N108Q-L145TN108Q-L145T cag-acccag-acc
L145TL145T accacc N108KN108K aagaag N108Q-Y149FN108Q-Y149F cag-ttccag-ttc
F148AF148A gcagca N108LN108L ctgctg  the  the
Y149FY149F ttcttc N108MN108M atgatg  the  the
L145AL145A gccgcc N108PN108P cctcct  the  the
L145DL145D gatgat N108RN108R agaaga  the  the
L145EL145E gaggag N108SN108S agcagc  the  the
L145FL145F ttcttc N108WN108W tggtgg  the  the
L145GL145G ggagga N108YN108Y tactac  the  the
L145HL145H caccac  the  the  the  the
TadA的氨基酸序列(SEQ ID NO:1)Amino acid sequence of TadA (SEQ ID NO: 1)
Figure PCTCN2022117592-appb-000001
Figure PCTCN2022117592-appb-000001
TadA的DNA序列(SEQ ID NO:2)DNA sequence of TadA (SEQ ID NO:2)
Figure PCTCN2022117592-appb-000002
Figure PCTCN2022117592-appb-000002
核定位信号序列的(SEQ ID NO:3)(SEQ ID NO:3) of nuclear localization signal sequence
Figure PCTCN2022117592-appb-000003
Figure PCTCN2022117592-appb-000003
实施例中使用的靶点及其序列如表2所示。The targets and their sequences used in the examples are shown in Table 2.
表2所用靶点及序列Table 2 Targets and sequences used
靶点名称target name 序列(5’-3’)Sequence (5'-3') SEQ ID NOSEQ ID NO
FANCF site1 FANCF site1 GGAATCCCTTCTGCAGCACCGGAATCCCTTCTGCAGCACC 44
EGFR-library-sg4EGFR-library-sg4 AAGATCAAAGTGCTGGGCTCAAGATCAAAGTGCTGGGCTC 55
HBG-sg1HBG-sg1 CTTGTCAAGGCTATTGGTCACTTGTCAAGGCTATTGGTCA 66
EMX1-sg2pEMX1-sg2p GACATCGATGTCCTCCCCATGACATCGATGTCCTCCCCAT 77
HBG-sg8HBG-sg8 CAGGACAAGGGAGGGAAGGACAGGACAAGGGAGGGAAGGA 88
ABE-site3ABE-site3 GTCAAGAAAGCAGAGACTGCGTCAAGAAAGCAGAGACTGC 99
ABE-site10ABE-site10 GAACATAAAGAATAGAATGAGAACATAAAGAATAGAATGA 1010
HEK-site7HEK-site7 GGAACACAAAGCATAGACTGGGAACACAAAGCATAGACTG 1111
ABE-site16ABE-site16 GGGAATAAATCATAGAATCCGGGAATAAATCATAGAATCC 1212
ABE-site17ABE-site17 GACAAAGAGGAAGAGAGACGGACAAAGAGGAAGAGAGACG 1313
ABE-site13ABE-site13 GAAGATAGAGAATAGACTGCGAAGATAGAGAATAGACTGC 1414
ABE-site8ABE-site8 GTAAACAAAGCATAGACTGAGTAAACAAAGCATAGACTGA 1515
实施例中使用的靶点的鉴定引物如表3所示。The identification primers of the targets used in the examples are shown in Table 3.
表3所用靶点的鉴定引物The identification primers of the targets used in Table 3
Figure PCTCN2022117592-appb-000004
Figure PCTCN2022117592-appb-000004
其中：F为正向引物，R为反向引物。Among them: F is the forward primer, R is the reverse primer.
实施例中使用的无缝克隆试剂盒为Vazyme ClonExpress MultiS One Step Cloning Kit，C113-01。The seamless cloning kit used in the examples is Vazyme ClonExpress MultiS One Step Cloning Kit, C113-01.
实施例中使用的HEK293T细胞为ATCC CRL-3216细胞系。The HEK293T cells used in the examples are ATCC CRL-3216 cell lines.
实施例中使用的质粒U6-sgRNA-EF1α-GFP的核苷酸序列如SEQ ID NO:40所示，其中，靶向靶序列的sgRNA的编码序列用连续的N表示。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.
U6-sgRNA-EF1α-GFP的核苷酸序列(SEQ ID NO:40)Nucleotide sequence (SEQ ID NO: 40) of U6-sgRNA-EF1α-GFP
Figure PCTCN2022117592-appb-000005
Figure PCTCN2022117592-appb-000005
Figure PCTCN2022117592-appb-000006
Figure PCTCN2022117592-appb-000006
实施例中测序的服务提供商为苏州金唯智生物科技有限公司。The service provider for sequencing in the examples is Suzhou Jinweizhi Biotechnology Co., Ltd.
实施例1Example 1
1.1质粒设计及构建1.1 Plasmid design and construction
1.1.1如图1所示，根据冷冻电镜捕捉ABE8e结合底物DNA的晶体结构，并根据该晶体结构，设计21个ABE8e的单点突变体(如表1所示)，同时设计了1个来自于人的内源性测试靶点FANCF site1(如表2所示)用于筛选评价。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 (as shown in Table 2) was used for screening evaluation.
1.1.2将21个ABE8e单点突变体按表1中的序列进行合成，以ABE8e为载体，之后进行无缝克隆组装，靶点即按表2合成两条oligo，正链加CACC，反链加上AAAC，连接至已经用BbsI酶切好的U6-sgRNA-EF1α-GFP上。1.1.2 Synthesize 21 single-point mutants of ABE8e according to the sequence in Table 1, use ABE8e as the carrier, and then perform seamless cloning and assembly. The target site is to synthesize two oligos according to Table 2, positive strand plus CACC, reverse strand Add AAAC and connect to U6-sgRNA-EF1α-GFP that has been digested with BbsI.
1.1.3将1.1.1与1.1.2中构建的质粒经sanger测序，确保序列完全正确。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细胞转染1.2 Cell transfection
第1天：用293T细胞铺种24孔板Day 1: Plating 24-well plates with 293T cells
(1)消化HEK293T细胞，按照2×105cell/孔接种96孔板。(1) Digest HEK293T cells and inoculate 96-well plates at 2×105 cells/well.
注意：细胞复苏后，一般需传代2次方可用于转染实验。Note: After recovery, the cells generally need to be subcultured twice before they can be used for transfection experiments.
第2天：转染Day 2: Transfection
(2)观察各孔细胞状态。(2) Observe the state of cells in each well.
注意：要求转染前细胞密度应为70％～90％，且状态正常。Note: It is required that the cell density before transfection should be 70%-90%, and the state should be normal.
(3)质粒转染量如下(以ABE8e作为对照)：(3) The amount of plasmid transfection is as follows (using ABE8e as a control):
ABE8e单点突变体：U6-sgRNA-EF1α-GFP＝750ng:250ngABE8e single point mutant: U6-sgRNA-EF1α-GFP=750ng:250ng
设置n＝3孔/组。Set n = 3 wells/group.
1.3基因组提取及扩增子文库的准备1.3 Genome extraction and amplicon library preparation
转染后72h，用天根细胞基因组提取试剂盒(DP304)提取细胞基因组DNA。之后用Hi-Tom Gene Editing Detection Kit(诺禾致源)的操作流程，设计相对应的鉴定引物(如表3所示)，即在正向引物5’端加上搭桥序列5’-ggagtgagtacggtgtgc-3’(SEQ ID NO:41)，反向引物5’端加上搭桥序列5’-gagttggatgctggatgg-3’(SEQ ID NO:42)，即得到一轮PCR产物，之后利用一轮PCR产物作为模板，进行二轮PCR，得到二轮PCR产物，之后混在一起进行切胶回收纯化后送公司进行测序。72h after transfection, the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Afterwards, 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 products, and then use the round of PCR products as templates , 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深度测序结果分析与统计1.4 Analysis and statistics of deep sequencing results
利用BE-analyzer网站(http://www.rgenome.net/be-analyzer/#！)分析深度测序结果，即统计A>G、C>T、C>G、C>A、Indel的比率，并用Graphpad Prism 9.1.0进行统计作图，如表4和图2～4所示。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 drawing, as shown in Table 4 and Figures 2-4.
1.5结果分析1.5 Result analysis
如表4和图2所示，根据Sanger结果，ABE8e的21个单点突变体中，ABE8e-L145T、ABE8e-L145Q、ABE8e-N108Q和ABE8e-F84T均显著降低旁观者A3的编辑，同时维持靶向碱基A4的编辑效率。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.
表4靶点FANCF site 1的编辑效率结果(单位，％)Table 4 Editing efficiency results of target FANCF site 1 (unit, %)
Figure PCTCN2022117592-appb-000007
Figure PCTCN2022117592-appb-000007
Figure PCTCN2022117592-appb-000008
Figure PCTCN2022117592-appb-000008
Figure PCTCN2022117592-appb-000009
Figure PCTCN2022117592-appb-000009
如图3所示，深度测序评价旁观者C6编辑，ABE8e产生的旁观者胞嘧啶编辑为45.2％(C>G+C>T+C>A)，而上述四种单点突变体产生的编辑效率分别仅为5.07％、3.67％、2.44％、2.93％，其中ABE8e-N108Q最高降低94.6％危害性胞嘧啶编辑。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%.
如图4所示，选择ABE8e-N108Q在另外四个内源性靶点(EGFR-library-sg4、HBG1-sg1、EMX1-sg2p、HBG-sg8)再次验证，结果表明：以ABE8e为对照，对于EGFR-library-sg4靶点，ABE8e-N108Q将旁观者胞嘧啶编辑从13.7％降低至3.13％，对于HBG-sg1靶点，旁观者胞嘧啶编辑从16.4％降低5.17％，对于EMX1-sg2P靶点，旁观者胞嘧啶编辑从14.5％降低1.9％，对于HBG-sg8靶点，旁观者胞嘧啶编辑从9.33％降低1.2％。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%.
综上，ABE8e-L145T、ABE8e-L145Q、ABE8e-N108Q和ABE8e-F84T均显著降低旁观者腺嘌呤编辑和旁观者胞嘧啶编辑。In summary, ABE8e-L145T, ABE8e-L145Q, ABE8e-N108Q and ABE8e-F84T all significantly reduced bystander adenine editing and bystander cytosine editing.
实施例2Example 2
为完全消除旁观者胞嘧啶编辑以及实现精准编辑单个A>G，设计本实施例。This embodiment is designed to completely eliminate bystander cytosine editing and to achieve precise editing of a single A>G.
2.1质粒设计及构建2.1 Plasmid design and construction
2.1.1基于实施例1的单点突变筛选结果，再次根据ABE8e的晶体结构，将潜在性影响底物结构的氨基酸位点进行组合突变合成，进行无缝克隆组装。同时设计2个富含poly A的内源性测试靶点ABE site10和ABE site3进行测试(如表2所示)，构建方法同1.1.2。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将2.1.1中构建的质粒经sanger测序，确保完全正确。2.1.2 Sanger sequenced the plasmid constructed in 2.1.1 to ensure that it is completely correct.
2.2细胞转染2.2 Cell transfection
第1天：用293T细胞铺种24孔板Day 1: Plating 24-well plates with 293T cells
(1)消化HEK293T细胞，按照2×10 5cell/孔接种96孔板。 (1) HEK293T cells were digested and inoculated into 96-well plates at 2×10 5 cells/well.
注意：细胞复苏后，一般需传代2次方可用于转染实验。Note: After recovery, the cells generally need to be subcultured twice before they can be used for transfection experiments.
第2天：转染Day 2: Transfection
(2)观察各孔细胞状态。(2) Observe the state of cells in each well.
注意：要求转染前细胞密度应为70％～90％，且状态正常。Note: It is required that the cell density before transfection should be 70%-90%, and the state should be normal.
(3)质粒转染量如下(以ABE8e作为对照)(3) The amount of plasmid transfection is as follows (using ABE8e as a control)
2.1中新构建的质粒：U6-sgRNA-EF1α-GFP＝750ng:250ngNewly constructed plasmid in 2.1: U6-sgRNA-EF1α-GFP=750ng:250ng
设置n＝3孔/组。Set n = 3 wells/group.
2.3基因组提取及扩增子文库的准备2.3 Genome extraction and amplicon library preparation
转染后72h，用天根细胞基因组提取试剂盒(DP304)提取细胞基因组DNA。之后用Hitom试剂盒的操作流程，设计相对应的鉴定引物(如表3所示)，即在正向引物5’端加上如SEQ ID NO:38所示的搭桥序列，反向引物5’端加上如SEQ ID NO:39所示的搭桥序列，即得到一轮PCR产物，之后利用一轮PCR产物作为模板，进行二轮PCR，得到二轮PCR产物，之后混在一起进行切胶回收纯化后送公司进行测序。72h after transfection, the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Afterwards, use the operation process of Hitom kit to design corresponding identification primers (as shown in table 3), that is, add the bridge sequence shown in SEQ ID NO:38 at the 5' end of the forward primer, and the reverse primer 5' Add the bridging sequence shown in SEQ ID NO: 39 to the end to obtain a round of PCR product, 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, and then mix them together for gel cutting, recovery and purification Sent to the company for sequencing.
2.4深度测序结果分析与统计2.4 Analysis and statistics of deep sequencing results
利用BE-analyzer网站(http://www.rgenome.net/be-analyzer/#！)分析深度测序结果，即统计A>G、C>T、C>G、C>A、Indel的比率,并用Graphpad Prism 9.1.0进行统计作图。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结果分析2.5 Result analysis
如图5所示，根据Sanger结果，在19个组合突变中，N108Q-L145T、N108Q-P29M、N108Q-P29W和N108Q-F84T显示了极窄的编辑窗口，靶向范围大约1～2个碱基。对于ABE site3靶点，ABE8e编辑范围为～5个碱基，而已报道的具备缩窄窗口潜能的F148A突变，编辑范围也有～4个碱基，ABE8e-N108Q编辑窗口为～3个碱基，而四种组合突变仅编辑1个碱基，且高效精准编辑A5。同样对于ABE site10靶点，ABE8e覆盖～7个碱基，ABE8e-F148A靶向区域为～4个碱基，对于单点突变ABE8e-N108Q，编辑范围依然为～3个碱基，N108Q-L145T和N108Q-P29M编辑范围为～2个碱基，同样高效催化第五位的A>G，N108Q-P29W和N108Q-F84T则精准编辑1个碱基A5，编辑活性仅部分降低。为便于描述，将ABE8e-N108Q命名为ABE9s，N108Q-L145T、N108Q-P29M、N108Q-P29W和N108Q-F84T依次命名为ABE9.1s、ABE9.2s、ABE9.3s和ABE9.4s。ABE9.1s、ABE9.2s、ABE9.3s和ABE9.4s的编辑窗口约为1～2个碱基，腺嘌呤编辑的选择性依次严格。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 ABE site3 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, and the editing window of ABE8e-N108Q is ~3 bases, while The four combined mutations only edited 1 base, and edited 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.
如图6所示，以ABE9.1s为例，以ABE site10以及新设计的内源性靶点HEK site7为评价靶点，再次评价组合突变的旁观者胞嘧啶编辑特征。结果显示，相较于ABE9s，ABE9.1s完全消除了危害性胞嘧啶编辑，同时缩窄窗口至1～2个碱基，并且偏好性编辑 A5/A6碱基。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.
综上，ABE8e的组合突变N108Q-L145T、N108Q-P29M、N108Q-P29W和N108Q-F84T可以精准编辑1～2个碱基，同时完全消除危害性胞嘧啶编辑。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.
实施例3Example 3
为对比ABE9.1s、ABE9.2s、ABE9.3s和ABE9.4s的工作特性，设计本实施例。This embodiment is designed to compare the working characteristics of ABE9.1s, ABE9.2s, ABE9.3s and ABE9.4s.
3.1质粒设计及构建3.1 Plasmid design and construction
3.1.1以ABE8e和ABE9s为对照，设计4个额外的靶点ABE-site16、ABE-site17、ABE-site13和ABE-site8(如表2所示)进行评价。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将3.1.1中构建的质粒经sanger测序，确保完全正确。3.1.2 Sanger sequenced the plasmid constructed in 3.1.1 to ensure that it is completely correct.
3.2细胞转染3.2 Cell transfection
第1天：用293T细胞铺种24孔板Day 1: Plating 24-well plates with 293T cells
(1)消化HEK293T细胞，按照2×10 5cell/孔接种96孔板。 (1) HEK293T cells were digested and inoculated into 96-well plates at 2×10 5 cells/well.
注意：细胞复苏后，一般需传代2次方可用于转染实验。Note: After recovery, the cells generally need to be subcultured twice before they can be used for transfection experiments.
第2天：转染Day 2: Transfection
(2)观察各孔细胞状态。(2) Observe the state of cells in each well.
注意：要求转染前细胞密度应为70％～90％，且状态正常。Note: It is required that the cell density before transfection should be 70%-90%, and the state should be normal.
(3)质粒转染量如下(以ABE8e作为对照)：(3) The amount of plasmid transfection is as follows (using ABE8e as a control):
3.1中新构建的质粒：U6-sgRNA-EF1α-GFP＝750ng:250ngNewly constructed plasmid in 3.1: U6-sgRNA-EF1α-GFP=750ng:250ng
设置n＝3孔/组。Set n = 3 wells/group.
3.3基因组提取及扩增子文库的准备3.3 Genome extraction and amplicon library preparation
转染后72h，用天根细胞基因组提取试剂盒(DP304)提取细胞基因组DNA。之后用Hitom试剂盒的操作流程，设计相对应的鉴定引物(如表3所示)，即在正向引物5’端加上如SEQ ID NO:38所示的搭桥序列，反向引物5’端加上如SEQ ID NO:39所示的搭桥序列，即得到一轮PCR产物，之后利用一轮PCR产物作为模板，进行二轮PCR，得到二轮PCR产物，之后混在一起进行切胶回收纯化后进行送公司进行测序。72h after transfection, the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Afterwards, use the operation process of Hitom kit to design corresponding identification primers (as shown in table 3), that is, add the bridge sequence shown in SEQ ID NO:38 at the 5' end of the forward primer, and the reverse primer 5' Add the bridging sequence shown in SEQ ID NO: 39 to the end to obtain a round of PCR product, 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, and then mix them together for gel cutting, recovery and purification Then send it to the company for sequencing.
3.4深度测序结果分析与统计3.4 Analysis and statistics of deep sequencing results
利用BE-analyzer网站(http://www.rgenome.net/be-analyzer/#！)分析深度测序结果，即统计A>G、C>T、C>G、C>A、Indel的比率,并用Graphpad Prism 9.1.0进行统计作图。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结果分析3.5 Result Analysis
如图7所示，在ABE site16位点，对应的ABE8e编辑窗口为～5个碱基，ABE9s可 覆盖～4个碱基，ABE9.1s、ABE9.2s、ABE9.3s、ABE9.4s仅编辑1～2个碱基；在ABE site13和ABE site13位点，ABE8e编辑范围为～5个碱基，ABE9s覆盖范围为3～4个碱基，而ABE9.1s、ABE9.2s、ABE9.3s和ABE9.4s中，除了ABE9.1s有轻微A7或者A4编辑外，其余三个变体均只编辑一个碱基。对于ABE site17位点，ABE8e编辑范围为～6个碱基，ABE9s编辑范围为～3个碱基，ABE9.1s和ABE9.2s主要编辑2个碱基(A5/A6)，ABE9.3s和ABE9.4s依然精确编辑单个碱基。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 ABE site13, 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 In ABE9.4s, except 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.
综上，ABE9s可轻微缩窄编辑范围，而ABE9.1s，ABE9.2s，ABE9.3s，ABE9.4s精准编辑1～2个碱基，同时保持较低的indel事件发生(如图8所示)。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 ).
实施例4Example 4
鉴于之前发现N108位点和L145位点部分单点突变和组合突变提高ABE编辑精度，我们尝试将这两个位点全饱和单点突变进一步挖掘潜在变体特性，设计本实施例。In view of the previous discovery that partial single-point mutations and combined mutations at N108 and L145 can improve the accuracy of ABE editing, we attempted to fully saturate these two single-point mutations to further explore potential variant characteristics and design this embodiment.
4.1质粒设计及构建4.1 Plasmid design and construction
4.1.1设计38个N108和L145饱和单点突变(如表1所示)，进行无缝克隆组装，以ABE8e和ABE9.1s为对照，设计1个人类内源性的靶点ABE-site13(如表2所示)进行评价，构建方法同1.1.2。4.1.1 Design 38 N108 and L145 saturation single-point mutations (as shown in Table 1), and perform seamless clone assembly. Using ABE8e and ABE9.1s as controls, design a human endogenous target ABE-site13 ( As shown in Table 2) to evaluate, the construction method is the same as 1.1.2.
4.1.2将3.1.1中构建的质粒经sanger测序，确保完全正确。4.1.2 Sanger sequence the plasmid constructed in 3.1.1 to ensure it is completely correct.
4.2细胞转染4.2 Cell transfection
第1天：用293T细胞铺种24孔板Day 1: Plating 24-well plates with 293T cells
(1)消化HEK293T细胞，按照2×105cell/孔接种96孔板。(1) Digest HEK293T cells and inoculate 96-well plates at 2×105 cells/well.
注意：细胞复苏后，一般需传代2次方可用于转染实验。Note: After recovery, the cells generally need to be subcultured twice before they can be used for transfection experiments.
第2天：转染Day 2: Transfection
(2)观察各孔细胞状态。(2) Observe the state of cells in each well.
注意：要求转染前细胞密度应为70％～90％，且状态正常。Note: It is required that the cell density before transfection should be 70%-90%, and the state should be normal.
(3)质粒转染量如下：(3) The amount of plasmid transfection is as follows:
4.1中新构建的质粒：U6-sgRNA-EF1α-GFP＝750ng:250ngNewly constructed plasmid in 4.1: U6-sgRNA-EF1α-GFP=750ng:250ng
设置n＝3孔/组。Set n = 3 wells/group.
4.3基因组提取及扩增子文库的准备4.3 Genome extraction and amplicon library preparation
转染后72h，用天根细胞基因组提取试剂盒(DP304)提取细胞基因组DNA。之后用Hitom试剂盒的操作流程，设计相对应的鉴定引物(如表3所示)，即在正向引物5’端加上如SEQ ID NO:38所示的搭桥序列，反向引物5’端加上如SEQ ID NO:39所示的 搭桥序列，即得到一轮PCR产物，之后利用一轮PCR产物作为模板，进行二轮PCR，得到二轮PCR产物，之后混在一起进行切胶回收纯化后进行送公司进行测序。72h after transfection, the genomic DNA of the cells was extracted with Tiangen Cell Genome Extraction Kit (DP304). Afterwards, use the operation process of Hitom kit to design corresponding identification primers (as shown in table 3), that is, add the bridge sequence shown in SEQ ID NO:38 at the 5' end of the forward primer, and the reverse primer 5' Add the bridging sequence shown in SEQ ID NO: 39 to the end to obtain a round of PCR product, 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, and then mix them together for gel cutting, recovery and purification Then send it to the company for sequencing.
4.4深度测序结果分析与统计4.4 Analysis and statistics of deep sequencing results
利用BE-analyzer网站(http://www.rgenome.net/be-analyzer/#！)分析深度测序结果，即统计A>G编辑效率,并用Graphpad Prism 9.1.0进行统计作图。Use the BE-analyzer website (http://www.rgenome.net/be-analyzer/#!) to analyze the deep sequencing results, that is, to count the A>G editing efficiency, and use Graphpad Prism 9.1.0 for statistical mapping.
4.5结果分析4.5 Result Analysis
如图9所示，在富含A的ABE site3位点，对应的ABE8e编辑活性窗口为A4-A7个碱基，ABE9.1仍主要精准编辑A5，而几乎所有N108位点突变相较于ABE8e均能有效缩窄编辑范围，且部分变体保持较高效率(如N108H、N108Q、N108S等)，而L145饱和突变中，L145D、L145E、L145G等突变相比于ABE8e也缩窄了编辑范围，意外的是L145W显著扩大了活性窗口(A4-A9)，A8和A9的编辑效率极大提高，相较ABE8e分别提高4.51倍和6.03倍。As shown in Figure 9, at the A-rich ABE site3 site, the corresponding ABE8e editing activity window is A4-A7 bases, ABE9.1 still mainly edits A5 precisely, and almost all N108 site mutations compared with ABE8e All can effectively narrow the editing range, and some variants maintain high efficiency (such as N108H, N108Q, N108S, etc.), and among L145 saturation mutations, mutations such as L145D, L145E, and L145G also narrow the editing range compared with ABE8e, Unexpectedly, L145W significantly expanded the active window (A4-A9), and the editing efficiency of A8 and A9 was greatly improved, which were 4.51 times and 6.03 times higher than ABE8e, respectively.
综上，N108位点和L145位点突变对于调控ABE编辑特性(窗口和活性等)起着至关重要的作用，如大部分的N108突变体和L145D、L145E、L145G等L145位点突变体，能够缩窄编辑窗口，提高编辑精度，而L145W却可以进一步扩大编辑范围，编辑效率极大提升，这使得之前靠近PAM区域无法编辑的腺嘌呤得以高效靶向，拓展了ABE的应用范围。In summary, N108 and L145 mutations play a crucial role in regulating ABE editing properties (window and activity, etc.), such as most N108 mutants and L145D, L145E, L145G and other L145 mutants, It can narrow the editing window and improve the editing accuracy, while L145W can further expand the editing range and greatly improve the editing efficiency, which enables efficient targeting of adenine that was previously uneditable near the PAM region and expands the application range of ABE.
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。Although the specific implementations of the present invention have been described above, those skilled in the art should understand that these are only examples, and various changes or changes can be made to these implementations without departing from the principle and essence of the present invention. Revise. Accordingly, the protection scope of the present invention is defined by the appended claims.

Claims (10)

  1. 一种腺嘌呤脱氨酶，其特征在于，所述腺嘌呤脱氨酶在包括如SEQ ID NO:1所示的氨基酸序列的第29位、第84位、第108位和第145位上存在一种或多种氨基酸差异。A kind of adenine deaminase, it is characterized in that, described adenine deaminase comprises the 29th, the 84th, the 108th and the 145th position of the amino acid sequence shown in SEQ ID NO:1 One or more amino acid differences.
  2. 如权利要求1所述的腺嘌呤脱氨酶，其特征在于，所述一种或多种氨基酸差异包括：Adenine deaminase as claimed in claim 1, is characterized in that, described one or more amino acid differences comprise:
    第108位氨基酸残基为Q、H、S、A、C、D、E、F、G、I、K、L、M、P、R、S、T、V、W或Y；或，Amino acid residue 108 is Q, H, S, A, C, D, E, F, G, I, K, L, M, P, R, S, T, V, W or Y; or,
    第145位氨基酸残基为T、A、C、D、E、F、G、H、I、K、M、N、P、Q、R、S、V、W或Y；或，Amino acid residue 145 is T, A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, V, W, or Y; or,
    第84位氨基酸残基为T；或，The 84th amino acid residue is T; or,
    第84位氨基酸残基为T，并且第108位氨基酸残基为Q；或，amino acid residue 84 is T and amino acid residue 108 is Q; or,
    第108位氨基酸残基为Q，并且第145位氨基酸残基为T；或，amino acid residue 108 is Q and amino acid residue 145 is T; or,
    第108位氨基酸残基为Q，并且第29位氨基酸残基为M；或，amino acid residue 108 is Q and amino acid residue 29 is M; or,
    第108位氨基酸残基为Q，并且第29位氨基酸残基为W；the 108th amino acid residue is Q, and the 29th amino acid residue is W;
    较佳地，所述腺嘌呤脱氨酶还包括核定位信号序列；所述核定位信号序列优选如SEQ ID NO:3所示。Preferably, the adenine deaminase also includes a nuclear localization signal sequence; the nuclear localization signal sequence is preferably as shown in SEQ ID NO:3.
  3. 一种腺嘌呤碱基编辑器，其特征在于，所述腺嘌呤碱基编辑器包括核酸酶和如权利要求1或2所述的腺嘌呤脱氨酶；An adenine base editor, characterized in that the adenine base editor comprises nuclease and the adenine deaminase according to claim 1 or 2;
    较佳地，所述核酸酶为Cas蛋白或其变体；Preferably, the nuclease is a Cas protein or a variant thereof;
    更佳地，所述Cas蛋白为酿酒酵母来源的spCas9、金黄色葡萄球菌来源的SaCas9、毛螺菌科细菌来源的LbCas12a或酸胺球菌属细菌来源的enAsCas12a；所述Cas蛋白变体为VQR-spCas9、VRER-spCas9、spRY、spNG、SaCas9-KKH或SaCas9-NG。More preferably, 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; the Cas protein variant is VQR- spCas9, VRER-spCas9, spRY, spNG, SaCas9-KKH, or SaCas9-NG.
  4. 一种融合蛋白，其特征在于，所述融合蛋白包含如权利要求1或2所述的腺嘌呤脱氨酶；A fusion protein, characterized in that, the fusion protein comprises the adenine deaminase as claimed in claim 1 or 2;
    较佳地，所述融合蛋白还包含核酸酶，所述核酸酶如权利要求3所定义。Preferably, the fusion protein further comprises a nuclease, and the nuclease is as defined in claim 3.
  5. 一种腺嘌呤碱基编辑系统，其特征在于，其包括：sgRNA和如权利要求3所述的腺嘌呤碱基编辑器；An adenine base editing system, characterized in that it comprises: sgRNA and the adenine base editor as claimed in claim 3;
    较佳地，所述sgRNA的靶序列如SEQ ID NO:4～15任一项所示。Preferably, the target sequence of the sgRNA is shown in any one of SEQ ID NO: 4-15.
  6. 一种药物组合物，其特征在于，所述药物组合物包括如权利要求1或2所述的腺嘌呤脱氨酶、如权利要求3所述的腺嘌呤碱基编辑器、如权利要求4所述的融合蛋白或 者如权利要求5所述的腺嘌呤碱基编辑系统；A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the adenine deaminase as claimed in claim 1 or 2, the adenine base editor as claimed in claim 3, the adenine base editor as claimed in claim 4 said fusion protein or the adenine base editing system as claimed in claim 5;
    较佳地，所述药物组合物还包括药学上可接受的载体。Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  7. 一种碱基编辑方法，其特征在于，所述碱基编辑方法包括：A base editing method, characterized in that the base editing method comprises:
    在靶细胞中表达如权利要求1或2所述的腺嘌呤脱氨酶、如权利要求3所述的腺嘌呤碱基编辑器、如权利要求4所述的融合蛋白或者如权利要求5所述的腺嘌呤碱基编辑系统，使所述靶细胞发生碱基编辑；Expressing the adenine deaminase according to claim 1 or 2, the adenine base editor according to claim 3, the fusion protein according to claim 4, or the fusion protein according to claim 5 in the target cell The adenine base editing system for base editing of the target cells;
    较佳地，所述碱基编辑方法还包括加入sgRNA；和/或，所述靶细胞的来源为哺乳动物或植物的分离的细胞系；Preferably, the base editing method also includes adding sgRNA; and/or, the source of the target cell is an isolated cell line of a mammal or a plant;
    更佳地，所述sgRNA的靶序列如SEQ ID NO:4～15任一项所示；和/或，所述分离的细胞系为293T细胞、HELA细胞、U2OS细胞、NIH3T3细胞或N2A细胞。More preferably, the target sequence of the sgRNA is shown in any one of SEQ ID NO: 4-15; and/or, the isolated cell line is 293T cells, HELA cells, U2OS cells, NIH3T3 cells or N2A cells.
  8. 一种如权利要求1或2所述的腺嘌呤脱氨酶、如权利要求3所述的腺嘌呤碱基编辑器、如权利要求4所述的融合蛋白或者如权利要求5所述的腺嘌呤碱基编辑系统在制备碱基编辑的药物或制备基因治疗的药物中的应用。An adenine deaminase as claimed in claim 1 or 2, an adenine base editor as claimed in claim 3, a fusion protein as claimed in claim 4 or an adenine as claimed in claim 5 The application of the base editing system in the preparation of base editing drugs or gene therapy drugs.
  9. 一种如权利要求1或2所述的腺嘌呤脱氨酶、如权利要求3所述的腺嘌呤碱基编辑器、如权利要求4所述的融合蛋白或者如权利要求5所述的腺嘌呤碱基编辑系统在构建动物模型和农作物育种中的应用。An adenine deaminase as claimed in claim 1 or 2, an adenine base editor as claimed in claim 3, a fusion protein as claimed in claim 4 or an adenine as claimed in claim 5 Application of base editing system in the construction of animal models and crop breeding.
  10. 一种如权利要求1或2所述的腺嘌呤脱氨酶、如权利要求3所述的腺嘌呤碱基编辑器、如权利要求4所述的融合蛋白或者如权利要求5所述的腺嘌呤碱基编辑系统在制备碱基编辑工具中的应用。An adenine deaminase as claimed in claim 1 or 2, an adenine base editor as claimed in claim 3, a fusion protein as claimed in claim 4 or an adenine as claimed in claim 5 Application of base editing system in preparation of base editing tools.
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