提高RNA碱基编辑mxABE精准靶向新策略
收稿日期: 2025-11-24
修回日期: 2026-01-28
网络出版日期: 2026-03-13
基金资助
广东省种业振兴行动项目(2024-XPY-00-015);科技创新2030-重大项目(2023ZD0404303)
A novel strategy to enhance precise targeting of the RNA base editor mxABE
Received date: 2025-11-24
Revised date: 2026-01-28
Online published: 2026-03-13
Supported by
Guangdong Province Seed Industry Revitalization Action Project(2024-XPY-00-015);Major Project of Science and Technology Innovation 2030(2023ZD0404303)
单核苷酸变异(single nucleotide variations，SNVs)是最常见的人类致病突变形式，而碱基编辑技术则为治疗这类致病性变异提供了理想的解决方案。RNA编辑技术因其作用可逆、无需永久改变基因组而规避了长期风险，且编辑器体积小巧，已成为当前基因治疗的热点。其中mini-dCas13X.1介导的RNA腺嘌呤碱基编辑(mxABE)系统展现出高效的RNA碱基编辑效率，但仍然存在旁观者编辑(bystander editing)效应导致的非靶向核苷酸编辑，构成了主要的脱靶风险。本研究通过在sgRNA序列中删除与非靶向腺苷相对的核苷酸，有效降低了mxABE系统的旁观者编辑效应。体外实验结果表明，该策略将旁观者编辑率成功控制在5%以下，并仍能维持约70%的高效目标位点编辑。在杜氏肌营养不良症(Duchenne muscular dystrophy，DMD)小鼠模型中，通过单次单个腺病毒(adeno-associated virus，AAV)注射递送mxABE系统后，结果显示胫骨前肌观察到显著的治疗效果，并成功消除了非靶向腺苷的旁观者编辑。本研究为mxABE的RNA编辑系统治疗单基因遗传疾病治疗提供了新的精准靶向策略。
刘谭, 蒋惠结, 王雪朗, 余瑶瑶, 王菲, 林佳佳, 杨化强 . 提高RNA碱基编辑mxABE精准靶向新策略[J]. 遗传, 2026 , 48(6) : 628 -637 . DOI: 10.16288/j.yczz.25-303
Single nucleotide variations (SNVs) represent the most common form of pathogenic mutations in humans, while base editing technology offers an ideal solution for treating such pathogenic variants. RNA editing has become a hotspot in current gene therapy due to its reversible action, which avoids long-term risks by not permanently altering the genome, and the compact size of the editors. Among these, the mini-dCas13X.1-mediated RNA adenine base editing (mxABE) system demonstrates highly efficient RNA base editing; however, it still suffers from non-target nucleotide editing caused by the bystander editing effect, which constitutes a major off-target risk. In this study, by deleting the nucleotide opposite the non-target adenosine in the sgRNA sequence, we effectively reduced the bystander editing effect of the mxABE system. In vitro results showed that this strategy successfully controlled the bystander editing rate below 5% while maintaining highly efficient on-target editing of approximately 70%. In a murine model of DMD (Duchenne muscular dystrophy), a single administration of AAV (adeno-associated virus)-delivered mxABE system demonstrated significant therapeutic efficacy in the tibialis anterior muscle, with successful elimination of off-target adenosine bystander editing. This study provides a novel precision-targeting strategy for treating monogenic genetic diseases using the mxABE RNA editing system.
| [1] | Rees HA, Liu DR. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet, 2018, 19(12):770-788. |
| [2] | Xiao QQ, Xu ZJ, Xue YY, Xu CL, Han L, Liu YH, Wang F, Zhang RZ, Han S, Wang X, Li GL, Li HW, Yang H, Shu YL. Rescue of autosomal dominant hearing loss by in vivo delivery of mini dCas13X-derived RNA base editor. Sci Transl Med, 2022, 14(654): eabn0449. |
| [3] | Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F. RNA editing with CRISPR- Cas13. Science, 2017, 358(6366): 1019-1027. |
| [4] | Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc, 2019, 14(10): 2986-3012. |
| [5] | Komor AC, Zhao KT, Packer MS, Gaudelli NM, Waterbury AL, Koblan LW, Kim YB, Badran AH, Liu DR. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity. Sci Adv, 2017, 3(8): eaao4774. |
| [6] | Li GL, Jin M, Li ZF, Xiao QQ, Lin JJ, Yang D, Liu YH, Wang X, Xie L, Ying WQ, Wang HQ, Zuo EW, Shi LY, Wang N, Chen WJ, Xu CL, Yang H. Mini-dCas13X- mediated RNA editing restores dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy. J Clin Invest, 2023, 133(3): e162809. |
| [7] | Lv J, Wang H, Cheng XT, Chen YX, Wang DQ, Zhang LL, Cao Q, Tang HH, Hu SW, Gao KY, Xun MZ, Wang JH, Wang ZJ, Zhu BY, Cui C, Gao ZW, Guo L, Yu S, Jiang LY, Yin YB, Zhang JJ, Chen B, Wang WQ, Chai RJ, Chen ZY, Li HW, Shu YL. AAV1-hOTOF gene therapy for autosomal recessive deafness 9: a single-arm trial. Lancet, 2024, 403(10441): 2317-2325. |
| [8] | Koblan LW, Doman JL, Wilson C, Levy JM, Tay T, Newby GA, Maianti JP, Raguram A, Liu DR. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol, 2018, 36(9): 843-846. |
| [9] | Lee SH, Wu J, Im D, Hwang GH, Jeong YK, Jiang H, Lee SJ, Jo DH, Goddard WA, Kim JH, Bae S. Bystander base editing interferes with visual function restoration in Leber congenital amaurosis. bioRxiv, 2024, doi: 10.1101/2024.10.23.619839. |
| [10] | Dharmadhikari AV, Abad MA, Khan S, Maroofian R, Sands TT, Ullah F, Samejima I, Wear MA, Moore KE, Kondakova E, Mitina N, Schaub T, Lee GK, Umandap CH, Berger SM, Iglesias AD, Popp B, Jamra RA, Gabriel H, Rentas S, Rippert AL, Izumi K, Conlin LK, Koboldt DC, Mosher TM, Hickey SE, Albert DVF, Norwood H, Lewanda AF, Dai HZ, Liu PF, Mitani T, Marafi D, Pehlivan D, Posey JE, Lippa N, Vena N, Heinzen EL, Goldstein DB, Mignot C, de Sainte Agathe JM, Al-Sannaa NA, Zamani M, Sadeghian S, Azizimalamiri R, Seifia T, Zaki MS, Abdel-Salam GMH, Abdel-Hamid M, Alabdi L, Alkuraya FS, Dawoud H, Lofty A, Bauer P, Zifarelli G, Afzal E, Zafar F, Efthymiou S, Gossett D, Towne MC, Yeneabat R, Wontakal SN, Aggarwal VS, Rosenfeld JA, Tarabykin V, Ohta S, Lupski JR, Houlden H, Earnshaw WC, Davis EE, Jeyaprakash AA, Liao J. RNA methyltransferase SPOUT1/CENP-32 links mitotic spindle organization with the neurodevelopmental disorder SpADMiSS. Nat Commun, 2025, 16(1): 1703. |
| [11] | Walker AS, Butt CM. Reusability Report: evaluating the performance of a meta-learning foundation model on predicting the antibacterial activity of natural products. Nat Mach Intell, 2026, 8(2): 270-275. |
| [12] | Valdez I, O’Connor I, Patel D, Gierer K, Harrington J, Ellis E, Caponetti SA, Sebra RP, Valley HC, Coote K, Mense M, Marro SG, Jiang T. A streamlined base editor engineering strategy to reduce bystander editing. Nat Commun, 2025, 16(1): 8115. |
| [13] | Ranzau BL, Rallapalli KL, Evanoff M, Paesani F, Komor AC. The wild-type tRNA adenosine deaminase enzyme TadA is capable of sequence-specific DNA base editing. Chembiochem, 2023, 24(16): e202200788. |
| [14] | Kim YB, Komor AC, Levy JM, Packer MS, Zhao KT, Liu DR. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat Biotechnol, 2017, 35(4): 371-376. |
| [15] | Chai AC, Cui M, Chemello F, Li H, Chen KN, Tan W, Atmanli A, McAnally JR, Zhang Y, Xu L, Liu N, Bassel-Duby R, Olson EN. Base editing correction of hypertrophic cardiomyopathy in human cardiomyocytes and humanized mice. Nat Med, 2023, 29(2): 401-411. |
| [16] | Huang SS, Zhang ZW, Tao WY, Liu Y, Li XY, Wang XL, Harati J, Wang PY, Huang XX, Lin CP. Broadening prime editing toolkits using RNA-Pol-II-driven engineered pegRNA. Mol Ther, 2022, 30(9): 2923-2932. |
| [17] | Zhang ZW, Tao WY, Huang SS, Sun WJ, Wang Y, Jiang W, Huang XX, Lin CP. Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities. Mol Ther Nucleic Acids, 2022, 29: 502-510. |
| [18] | Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol, 2020, 38(7): 883-891. |
| [19] | Huang TP, Zhao KT, Miller SM, Gaudelli NM, Oakes BL, Fellmann C, Savage DF, Liu DR. Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors. Nat Biotechnol, 2019, 37(6): 626-631. |
| [20] | Gaudelli NM, Lam DK, Rees HA, Solá-Esteves NM, Barrera LA, Born DA, Edwards A, Gehrke JM, Lee SJ, Liquori AJ, Murray R, Packer MS, Rinaldi C, Slaymaker IM, Yen J, Young LE, Ciaramella G. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat Biotechnol, 2020, 38(7): 892-900. |
| [21] | Hu JH, Miller SM, Geurts MH, Tang WX, Chen LW, Sun N, Zeina CM, Gao X, Rees HA, Lin Z, Liu DR. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature, 2018, 556(7699): 57-63. |
| [22] | Xiong XY, Liu KH, Li ZX, Xia FN, Ruan XM, He XL, Li JF. Split complementation of base editors to minimize off-target edits. Nat Plants, 2023, 9(11): 1832-1847. |
| [23] | Liu ZZ, Yang LZ, Yang YH, Li JT, Chen Z, Guo CT, Guo QH, Li QX, Zhao DY, Hu XM, Gao F, Guo YX. ABE- mediated cardiac gene silencing via single AAVs requires DNA accessibility. Circ Res, 2025, 136(3): 318-320. |
| [24] | Davis JR, Banskota S, Levy JM, Newby GA, Wang X, Anzalone AV, Nelson AT, Chen PJ, Hennes AD, An MR, Roh H, Randolph PB, Musunuru K, Liu DR. Efficient prime editing in mouse brain, liver and heart with dual AAVs. Nat Biotechnol, 2024, 42(2): 253-264. |
/
| 〈 |
|
〉 |
www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:
