2024 05 v.40 996-1009
靶向HIV-1的Cas13a/crRNA的快速筛选和抗病毒活力测定
基金项目(Foundation):
国家科技重大专项（项目号：2017ZX10202102-007）,题目：艾滋病和病毒性肝炎等重大传染病防治;;
湖北省自然科学基金计划面上类项目（项目号：2019CFB529）,题目：潜伏性病毒激活剂联合CAR-T细胞清除体内HIV病毒“储藏库”的研究~~
邮箱(Email):
guchaoji-ang@wust.edu.cn;
DOI:
10.13242/j.cnki.bingduxuebao.004496
中文作者单位:
武汉科技大学生命科学与健康学院;
摘要(Abstract):
目前基于规律成簇的间隔短回文重复（Clustered regularly interspaced short palindromic repeats, CRISPR）/CRISPR相关蛋白（Cas）系统的基因治疗在遗传病、肿瘤及感染性疾病等领域均取得重大突破，其中Cas13a属于VI型CRISPR系统，Cas13a蛋白在HIV感染细胞内crRNA的引导下定位目标RNA，切割核酸，同时激活Cas13a蛋白的附属效应（Collateral effect）可高效切割细胞内非特异性单链RNA，从而诱导感染细胞凋亡，进一步破坏病毒库，是最有可能功能性治愈HIV的策略。本研究旨在筛选靶向HIV-1的高灵敏度crRNA，进一步探索组合不同crRNA对HIV-1的抑制效果，并在细胞转染水平证明Cas13a/crRNA系统的附属切割活性及其诱导细胞凋亡。在LANL数据库中比对HIV-1各亚型毒株序列，获得各功能蛋白高度保守区。同时，结合HIV数据库中RNA的剪接供体及受体位点信息；然后利用MPRDOCK软件进行crRNA与LwCas13a的分子对接并计算结合复合物的自由能大小选定一组crRNA池；再以荧光标记RNA探针与LwCas13a蛋白组成可视化检测体系，以FAM荧光信号强弱确定了最有效的crRNA。构建LwCas13a和crRNA的共表达质粒，通过共转染HIV全长感染性克隆质粒和含单个或多个crRNA组合的LwCas13a的表达质粒到HEK293T细胞中，根据转染细胞及其上清再感染细胞中萤光素酶和病毒结构蛋白RNA表达水平来判定CRISPR-Cas13a系统对HIV基因表达的抑制效率。初步筛选出优选的27条crRNA并结合切割荧光强弱从中筛选出8条高灵敏度crRNA。细胞共转染实验表明，靶向病毒结构基因的单个crRNA,crGag2,crPol1,crA53能降低HIV-1子代病毒RNA表达降低了43.2%～58.3%，同样，再次感染实验也证明子代病毒活性滴度降低了38%～74.6%。进一步的结果证明，4个crRNA(crD15,crA53,crGag2,crPol1)联用比单个crRNA表达降低了86.4%，比两个crRNAs降低了64.9%，同时在共转染HEK293T细胞中观察到Cas13a蛋白的附属切割活性及其诱导细胞凋亡。我们建立的CRISPR/Cas13a系统能够显著抑制HIV病毒复制和子代病毒产生并诱导细胞凋亡，为HIV的治疗提供了一种新的工具。
关键词(KeyWords):
HIV-1;;CRISPR/Cas13a;;分子对接;;RNA编辑
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参考文献
[1] Collaboration A T C. Survival of HIV-positive patients starting antiretroviral therapy between 1996 and 2013:a collaborative analysis of cohort studies[J]. Lancet HIV,2017, 4(8):e349-e356. DOI:10.1016/S2352-3018(17)30066-8.
[2] Siliciano J D, Kajdas J, Finzi D, et al. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+T cells[J]. Nat Med, 2003, 9(6):727-728. DOI:10.1038/nm880.
[3] Wagner T A, McLaughlin S, Garg K, et al. HIV latency. Proliferation of cells with HIV integrated into cancer genes contributes to persistent infection[J].Science, 2014, 345(6196):570-573. DOI:10.1126/science.1256304.
[4] Maldarelli F, Wu X, Su L, et al. HIV latency. Specific HIV integration sites are linked to clonal expansion and persistence of infected cells[J]. Science, 2014, 345(6193):179-183. DOI:10.1126/science.1254194.
[5] Pennings P S. HIV Drug Resistance:Problems and Perspectives[J]. Infect Dis Rep, 2013, 5(Suppl 1):e5.DOI:10.4081/idr.2013.s1.e5.
[6] Kordelas L, Verheyen J, Beelen D W, et al. Shift of HIV tropism in stem-cell transplantation with CCR5Delta32 mutation[J]. N Engl J Med, 2014, 371(9):880-882. DOI:10.1056/NEJMc1405805.
[7] Gupta R K, Abdul-Jawad S, McCoy L E, et al. HIV-1remission following CCR5Delta32/Delta32haematopoietic stem-cell transplantation[J]. Nature,2019, 568(7751):244-248. DOI:10.1038/s41586-019-1027-4.
[8] Xu L, Wang J, Liu Y, et al. CRISPR-Edited Stem Cells in a Patient with HIV and Acute Lymphocytic Leukemia[J]. N Engl J Med, 2019, 381(13):1240-1247. DOI:10.1056/NEJMoa1817426.
[9] Kessing C F, Nixon C C, Li C, et al. In Vivo Suppression of HIV Rebound by Didehydro-Cortistatin A, a"Block-and-Lock"Strategy for HIV-1 Treatment[J]. Cell Rep, 2017, 21(3):600-611. DOI:10.1016/j.celrep.2017.09.080.
[10]Borducchi E N, Cabral C, Stephenson K E, et al.Ad26/MVA therapeutic vaccination with TLR7stimulation in SIV-infected rhesus monkeys[J]. Nature,2016, 540(7632):284-287. DOI:10.1038/nature20583.
[11]Tebas P, Jadlowsky J K, Shaw P A, et al. CCR5-edited CD4+T cells augment HIV-specific immunity to enable post-rebound control of HIV replication[J]. J Clin Invest, 2021, 131(7). DOI:10.1172/JCI144486.
[12]Roberts M R, Qin L, Zhang D, et al. Targeting of human immunodeficiency virus-infected cells by CD8+T lymphocytes armed with universal T-cell receptors[J]. Blood, 1994, 84(9):2878-2889. DOI:10.1182/blood.V84.9.2878.bloodjournal8492878.
[13]Frangoul H, Altshuler D, Cappellini M D, et al.CRISPR-Cas9 Gene Editing for Sickle Cell Disease and beta-Thalassemia[J]. N Engl J Med, 2021, 384(3):252-260. DOI:10.1056/NEJMoa2031054.
[14]Yin D, Ling S, Wang D, et al. Targeting herpes simplex virus with CRISPR-Cas9 cures herpetic stromal keratitis in mice[J]. Nat Biotechnol, 2021, 39(5):567-577. DOI:10.1038/s41587-020-00781-8.
[15]Lee R G, Mazzola A M, Braun M C, et al. Efficacy and Safety of an Investigational Single-Course CRISPR Base-Editing Therapy Targeting PCSK9 in Nonhuman Primate and Mouse Models[J]. Circulation, 2023, 147(3):242-253.DOI:10.1161/CIRCULATIONAHA.122.062132.
[16]Maeder M L, Stefanidakis M, Wilson C J, et al.Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10[J]. Nat Med, 2019, 25(2):229-233. DOI:10.1038/s41591-018-0327-9.
[17]Mancuso P, Chen C, Kaminski R, et al. CRISPR based editing of SIV proviral DNA in ART treated non-human primates[J]. Nat Commun, 2020, 11(1):6065. DOI:10.1038/s41467-020-19821-7.
[18]Abudayyeh O O, Gootenberg J S, Konermann S, et al.C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector[J]. Science, 2016,353(6299):aaf5573. DOI:10.1126/science.aaf5573.
[19]East-Seletsky A, O'Connell M R, Burstein D, et al.RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes[J]. Mol Cell, 2017, 66(3):373-383 e373. DOI:10.1016/j.molcel.2017.04.008.
[20]Liu L, Li X, Wang J, et al. Two Distant Catalytic Sites Are Responsible for C2c2 RNase Activities[J]. Cell,2017, 168(1-2):121-134 e112. DOI:10.1016/j.cell.2016.12.031.
[21]Gootenberg J S, Abudayyeh O O, Lee J W, et al.Nucleic acid detection with CRISPR-Cas13a/C2c2[J].Science, 2017, 356(6336):438-442. DOI:10.1126/science.aam9321
[22]Ackerman C M, Myhrvold C, Thakku S G, et al.Massively multiplexed nucleic acid detection with Cas13[J]. Nature, 2020, 582(7811):277-282. DOI:10.1038/s41586-020-2279-8.
[23]Gootenberg J S, Abudayyeh O O, Kellner M J, et al.Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6[J]. Science, 2018, 360(6387):439-444. DOI:10.1126/science.aaq0179.
[24]Abudayyeh O O, Gootenberg J S, Essletzbichler P, et al. RNA targeting with CRISPR-Cas13[J]. Nature,2017, 550(7675):280-284. DOI:10.1038/nature24049.
[25]Konermann S, Lotfy P, Brideau N J, et al.Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors[J]. Cell, 2018, 173(3):665-676 e614. DOI:10.1016/j.cell.2018.02.033.
[26]Aman R, Ali Z, Butt H, et al. RNA virus interference via CRISPR/Cas13a system in plants[J]. Genome Biol,2018, 19(1):1. DOI:10.1186/s13059-017-1381-1.
[27]Jing X, Xie B, Chen L, et al. Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing[J]. Nucleic Acids Res, 2018, 46(15):e90. DOI:10.1093/nar/gky433.
[28]Kushawah G, Hernandez-Huertas L, Abugattas-Nunez Del Prado J, et al. CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos[J]. Dev Cell,2020, 54(6):805-817 e807. DOI:10.1016/j.devcel.2020.07.013.
[29]Abbott T R, Dhamdhere G, Liu Y, et al. Development of CRISPR as an Antiviral Strategy to Combat SARSCoV-2 and Influenza[J]. Cell, 2020, 181(4):865-876e812. DOI:10.1016/j.cell.2020.04.020.
[30]Li S, Li X, Xue W, et al. Screening for functional circular RNAs using the CRISPR-Cas13 system[J]. Nat Methods, 2021, 18(1):51-59. DOI:10.1038/s41592-020-01011-4.
[31]Xu C, Zhou Y, Xiao Q, et al. Programmable RNA editing with compact CRISPR-Cas13 systems from uncultivated microbes[J]. Nat Methods, 2021, 18(5):499-506. DOI:10.1038/s41592-021-01124-4.
[32]Li H, Wang S, Dong X, et al. CRISPR-Cas13a Cleavage of Dengue Virus NS3 Gene Efficiently Inhibits Viral Replication[J]. Mol Ther Nucleic Acids, 2020, 19(2162-2531(Print)):1460-1469. DOI:10.1016/j.omtn.2020.01.028.
[33]Yin L, Zhao F, Sun H, et al. CRISPR-Cas13a Inhibits HIV-1 Infection[J]. Mol Ther Nucleic Acids, 2020, 21(2162-2531(Print)):147-155. DOI:10.1016/j.omtn.2020.05.030.
[34]Nguyen H, Wilson H, Jayakumar S, et al. Efficient Inhibition of HIV Using CRISPR/Cas13d Nuclease System[J]. Viruses, 2021, 13(9):1850. DOI:10.3390/v13091850.
[35]Nunes-Alves C. Viral infection:Seeding the HIV-1reservoir[J]. Nat Rev Microbiol, 2014, 12(9):594.DOI:10.1038/nrmicro3342.
[36]Whitney J B, Hill A L, Sanisetty S, et al. Rapid seeding of the viral reservoir prior to SIV viraemia in rhesus monkeys[J]. Nature, 2014, 512(7512):74-77.DOI:10.1038/nature13594.
[37]Landovitz R J, Scott H, Deeks S G. Prevention,treatment and cure of HIV infection[J]. Nat Rev Microbiol, 2023, 21(10):657-670. DOI:10.1038/s41579-023-00914-1.
[38]Gaebler C, Nogueira L, Stoffel E, et al. Prolonged viral suppression with anti-HIV-1 antibody therapy[J].Nature, 2022, 606(7913):368-374. DOI:10.1038/s41586-022-04597-1.
[39]Niessl J, Baxter A E, Mendoza P, et al. Combination anti-HIV-1 antibody therapy is associated with increased virus-specific T cell immunity[J]. Nat Med, 2020, 26(2):222-227. DOI:10.1038/s41591-019-0747-1.
[40]Gunst J D, Pahus M H, Rosas-Umbert M, et al. Early intervention with 3BNC117 and romidepsin at antiretroviral treatment initiation in people with HIV-1:a phase 1b/2a, randomized trial[J]. Nat Med, 2022, 28(11):2424-2435. DOI:10.1038/s41591-022-02023-7.
[41]Yukl S A, Boritz E, Busch M, et al. Challenges in detecting HIV persistence during potentially curative interventions:a study of the Berlin patient[J]. PLoS Pathog, 2013, 9(5):e1003347. DOI:10.1371/journal.ppat.1003347.
[42]Wu Y, Jin W, Wang Q, et al. Precise editing of FGFR3-TACC3 fusion genes with CRISPR-Cas13a in glioblastoma[J]. Mol Ther, 2021, 29(11):3305-3318.DOI:10.1016/j.ymthe.2021.07.002.
[43]Tong H, Huang J, Xiao Q, et al. High-fidelity Cas13variants for targeted RNA degradation with minimal collateral effects[J]. Nat Biotechnol, 2023, 41(1):108-119. DOI:10.1038/s41587-022-01419-7.
[44]Wang D, Zhang F, Gao G. CRISPR-Based Therapeutic Genome Editing:Strategies and In Vivo Delivery by AAV Vectors[J]. Cell, 2020, 181(1):136-150. DOI:10.1016/j.cell.2020.03.023.
[45]Wang D, Tai P W L, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery[J]. Nat Rev Drug Discov, 2019, 18(5):358-378. DOI:10.1038/s41573-019-0012-9.
[46]Cecchin R, Troyer Z, Witwer K, et al. Extracellular vesicles:The next generation in gene therapy delivery[J]. Mol Ther, 2023, 31(5):1225-1230. DOI:10.1016/j.ymthe.2023.01.021.
[2] Siliciano J D, Kajdas J, Finzi D, et al. Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+T cells[J]. Nat Med, 2003, 9(6):727-728. DOI:10.1038/nm880.
[3] Wagner T A, McLaughlin S, Garg K, et al. HIV latency. Proliferation of cells with HIV integrated into cancer genes contributes to persistent infection[J].Science, 2014, 345(6196):570-573. DOI:10.1126/science.1256304.
[4] Maldarelli F, Wu X, Su L, et al. HIV latency. Specific HIV integration sites are linked to clonal expansion and persistence of infected cells[J]. Science, 2014, 345(6193):179-183. DOI:10.1126/science.1254194.
[5] Pennings P S. HIV Drug Resistance:Problems and Perspectives[J]. Infect Dis Rep, 2013, 5(Suppl 1):e5.DOI:10.4081/idr.2013.s1.e5.
[6] Kordelas L, Verheyen J, Beelen D W, et al. Shift of HIV tropism in stem-cell transplantation with CCR5Delta32 mutation[J]. N Engl J Med, 2014, 371(9):880-882. DOI:10.1056/NEJMc1405805.
[7] Gupta R K, Abdul-Jawad S, McCoy L E, et al. HIV-1remission following CCR5Delta32/Delta32haematopoietic stem-cell transplantation[J]. Nature,2019, 568(7751):244-248. DOI:10.1038/s41586-019-1027-4.
[8] Xu L, Wang J, Liu Y, et al. CRISPR-Edited Stem Cells in a Patient with HIV and Acute Lymphocytic Leukemia[J]. N Engl J Med, 2019, 381(13):1240-1247. DOI:10.1056/NEJMoa1817426.
[9] Kessing C F, Nixon C C, Li C, et al. In Vivo Suppression of HIV Rebound by Didehydro-Cortistatin A, a"Block-and-Lock"Strategy for HIV-1 Treatment[J]. Cell Rep, 2017, 21(3):600-611. DOI:10.1016/j.celrep.2017.09.080.
[10]Borducchi E N, Cabral C, Stephenson K E, et al.Ad26/MVA therapeutic vaccination with TLR7stimulation in SIV-infected rhesus monkeys[J]. Nature,2016, 540(7632):284-287. DOI:10.1038/nature20583.
[11]Tebas P, Jadlowsky J K, Shaw P A, et al. CCR5-edited CD4+T cells augment HIV-specific immunity to enable post-rebound control of HIV replication[J]. J Clin Invest, 2021, 131(7). DOI:10.1172/JCI144486.
[12]Roberts M R, Qin L, Zhang D, et al. Targeting of human immunodeficiency virus-infected cells by CD8+T lymphocytes armed with universal T-cell receptors[J]. Blood, 1994, 84(9):2878-2889. DOI:10.1182/blood.V84.9.2878.bloodjournal8492878.
[13]Frangoul H, Altshuler D, Cappellini M D, et al.CRISPR-Cas9 Gene Editing for Sickle Cell Disease and beta-Thalassemia[J]. N Engl J Med, 2021, 384(3):252-260. DOI:10.1056/NEJMoa2031054.
[14]Yin D, Ling S, Wang D, et al. Targeting herpes simplex virus with CRISPR-Cas9 cures herpetic stromal keratitis in mice[J]. Nat Biotechnol, 2021, 39(5):567-577. DOI:10.1038/s41587-020-00781-8.
[15]Lee R G, Mazzola A M, Braun M C, et al. Efficacy and Safety of an Investigational Single-Course CRISPR Base-Editing Therapy Targeting PCSK9 in Nonhuman Primate and Mouse Models[J]. Circulation, 2023, 147(3):242-253.DOI:10.1161/CIRCULATIONAHA.122.062132.
[16]Maeder M L, Stefanidakis M, Wilson C J, et al.Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10[J]. Nat Med, 2019, 25(2):229-233. DOI:10.1038/s41591-018-0327-9.
[17]Mancuso P, Chen C, Kaminski R, et al. CRISPR based editing of SIV proviral DNA in ART treated non-human primates[J]. Nat Commun, 2020, 11(1):6065. DOI:10.1038/s41467-020-19821-7.
[18]Abudayyeh O O, Gootenberg J S, Konermann S, et al.C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector[J]. Science, 2016,353(6299):aaf5573. DOI:10.1126/science.aaf5573.
[19]East-Seletsky A, O'Connell M R, Burstein D, et al.RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes[J]. Mol Cell, 2017, 66(3):373-383 e373. DOI:10.1016/j.molcel.2017.04.008.
[20]Liu L, Li X, Wang J, et al. Two Distant Catalytic Sites Are Responsible for C2c2 RNase Activities[J]. Cell,2017, 168(1-2):121-134 e112. DOI:10.1016/j.cell.2016.12.031.
[21]Gootenberg J S, Abudayyeh O O, Lee J W, et al.Nucleic acid detection with CRISPR-Cas13a/C2c2[J].Science, 2017, 356(6336):438-442. DOI:10.1126/science.aam9321
[22]Ackerman C M, Myhrvold C, Thakku S G, et al.Massively multiplexed nucleic acid detection with Cas13[J]. Nature, 2020, 582(7811):277-282. DOI:10.1038/s41586-020-2279-8.
[23]Gootenberg J S, Abudayyeh O O, Kellner M J, et al.Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6[J]. Science, 2018, 360(6387):439-444. DOI:10.1126/science.aaq0179.
[24]Abudayyeh O O, Gootenberg J S, Essletzbichler P, et al. RNA targeting with CRISPR-Cas13[J]. Nature,2017, 550(7675):280-284. DOI:10.1038/nature24049.
[25]Konermann S, Lotfy P, Brideau N J, et al.Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors[J]. Cell, 2018, 173(3):665-676 e614. DOI:10.1016/j.cell.2018.02.033.
[26]Aman R, Ali Z, Butt H, et al. RNA virus interference via CRISPR/Cas13a system in plants[J]. Genome Biol,2018, 19(1):1. DOI:10.1186/s13059-017-1381-1.
[27]Jing X, Xie B, Chen L, et al. Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing[J]. Nucleic Acids Res, 2018, 46(15):e90. DOI:10.1093/nar/gky433.
[28]Kushawah G, Hernandez-Huertas L, Abugattas-Nunez Del Prado J, et al. CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos[J]. Dev Cell,2020, 54(6):805-817 e807. DOI:10.1016/j.devcel.2020.07.013.
[29]Abbott T R, Dhamdhere G, Liu Y, et al. Development of CRISPR as an Antiviral Strategy to Combat SARSCoV-2 and Influenza[J]. Cell, 2020, 181(4):865-876e812. DOI:10.1016/j.cell.2020.04.020.
[30]Li S, Li X, Xue W, et al. Screening for functional circular RNAs using the CRISPR-Cas13 system[J]. Nat Methods, 2021, 18(1):51-59. DOI:10.1038/s41592-020-01011-4.
[31]Xu C, Zhou Y, Xiao Q, et al. Programmable RNA editing with compact CRISPR-Cas13 systems from uncultivated microbes[J]. Nat Methods, 2021, 18(5):499-506. DOI:10.1038/s41592-021-01124-4.
[32]Li H, Wang S, Dong X, et al. CRISPR-Cas13a Cleavage of Dengue Virus NS3 Gene Efficiently Inhibits Viral Replication[J]. Mol Ther Nucleic Acids, 2020, 19(2162-2531(Print)):1460-1469. DOI:10.1016/j.omtn.2020.01.028.
[33]Yin L, Zhao F, Sun H, et al. CRISPR-Cas13a Inhibits HIV-1 Infection[J]. Mol Ther Nucleic Acids, 2020, 21(2162-2531(Print)):147-155. DOI:10.1016/j.omtn.2020.05.030.
[34]Nguyen H, Wilson H, Jayakumar S, et al. Efficient Inhibition of HIV Using CRISPR/Cas13d Nuclease System[J]. Viruses, 2021, 13(9):1850. DOI:10.3390/v13091850.
[35]Nunes-Alves C. Viral infection:Seeding the HIV-1reservoir[J]. Nat Rev Microbiol, 2014, 12(9):594.DOI:10.1038/nrmicro3342.
[36]Whitney J B, Hill A L, Sanisetty S, et al. Rapid seeding of the viral reservoir prior to SIV viraemia in rhesus monkeys[J]. Nature, 2014, 512(7512):74-77.DOI:10.1038/nature13594.
[37]Landovitz R J, Scott H, Deeks S G. Prevention,treatment and cure of HIV infection[J]. Nat Rev Microbiol, 2023, 21(10):657-670. DOI:10.1038/s41579-023-00914-1.
[38]Gaebler C, Nogueira L, Stoffel E, et al. Prolonged viral suppression with anti-HIV-1 antibody therapy[J].Nature, 2022, 606(7913):368-374. DOI:10.1038/s41586-022-04597-1.
[39]Niessl J, Baxter A E, Mendoza P, et al. Combination anti-HIV-1 antibody therapy is associated with increased virus-specific T cell immunity[J]. Nat Med, 2020, 26(2):222-227. DOI:10.1038/s41591-019-0747-1.
[40]Gunst J D, Pahus M H, Rosas-Umbert M, et al. Early intervention with 3BNC117 and romidepsin at antiretroviral treatment initiation in people with HIV-1:a phase 1b/2a, randomized trial[J]. Nat Med, 2022, 28(11):2424-2435. DOI:10.1038/s41591-022-02023-7.
[41]Yukl S A, Boritz E, Busch M, et al. Challenges in detecting HIV persistence during potentially curative interventions:a study of the Berlin patient[J]. PLoS Pathog, 2013, 9(5):e1003347. DOI:10.1371/journal.ppat.1003347.
[42]Wu Y, Jin W, Wang Q, et al. Precise editing of FGFR3-TACC3 fusion genes with CRISPR-Cas13a in glioblastoma[J]. Mol Ther, 2021, 29(11):3305-3318.DOI:10.1016/j.ymthe.2021.07.002.
[43]Tong H, Huang J, Xiao Q, et al. High-fidelity Cas13variants for targeted RNA degradation with minimal collateral effects[J]. Nat Biotechnol, 2023, 41(1):108-119. DOI:10.1038/s41587-022-01419-7.
[44]Wang D, Zhang F, Gao G. CRISPR-Based Therapeutic Genome Editing:Strategies and In Vivo Delivery by AAV Vectors[J]. Cell, 2020, 181(1):136-150. DOI:10.1016/j.cell.2020.03.023.
[45]Wang D, Tai P W L, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery[J]. Nat Rev Drug Discov, 2019, 18(5):358-378. DOI:10.1038/s41573-019-0012-9.
[46]Cecchin R, Troyer Z, Witwer K, et al. Extracellular vesicles:The next generation in gene therapy delivery[J]. Mol Ther, 2023, 31(5):1225-1230. DOI:10.1016/j.ymthe.2023.01.021.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004496
中图分类号:R512.91
引用信息:
[1]王涛,夏学娇,马妍等.靶向HIV-1的Cas13a/crRNA的快速筛选和抗病毒活力测定[J].病毒学报,2024,40(05):996-1009.DOI:10.13242/j.cnki.bingduxuebao.004496.
基金信息:
国家科技重大专项（项目号：2017ZX10202102-007）,题目：艾滋病和病毒性肝炎等重大传染病防治;; 湖北省自然科学基金计划面上类项目（项目号：2019CFB529）,题目：潜伏性病毒激活剂联合CAR-T细胞清除体内HIV病毒“储藏库”的研究~~
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