dCas9基因激活系统在HIV体外扩增检测中的应用
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摘要
目的改进和提高HIV体外扩增检测的灵敏度,缩短检测时间。方法 1)用Broad Institute gRNA设计工具选取HIV的长末端重复序列(LTR)段设计20条gRNA(引导RNA)。2)用含荧光素酶报告的基因HIV(HIV-Luc)感染具有dCas9基因激活系统的Jurkat6465细胞系构建Jurkat6465-Luc细胞系,并将20条gRNA置于Jurkat6465-Luc细胞系中筛选出激活能力筛选,选取效率最高的gRNA-L,gRNA-L与dCas9基因激活系统包装成慢病毒感染Jurkat细胞系,建立可对HIV序列高效扩增的工作细胞系Jurkat6465-L。3)用HIV-Luc感染Jurkat细胞系构建HIV潜伏测试细胞系,设定其潜伏频率为1∶500,并与工作细胞Jurkat6465-L置于细胞培养箱中共同培养7和14 d观察工作细胞对HIV潜伏细胞系的激活能力。4)使用HIV激活剂SAHA和dCas9基因激活系统分别作用于设定潜伏频率为1∶500的潜伏细胞,分别比较对潜伏HIV的激活能力。结果首先,测定荧光素酶活性筛选出的2条具有高效HIV扩增能力的gRNA-L和gRNA-O,二者及对照组促进Jurkat6465-Luc的扩增效力(荧光素酶活性强度)(n=3,pg/mL),6 d分别为:85 530±979.2 vs 96 925±2 759 vs 9 876±437(P<0.01);选取gRNA-L与dCas9基因激活系统构建的Jurkat6465L细胞系作为工作细胞,测试设定潜伏频率为1/500的HIV潜伏测试细胞,在培养7、14 d激活HIV潜伏测试细胞的频率测定分别1/864和1/755,比培养14 d用SAHA激活HIV潜伏模型细胞频率1/1 180更接近设定潜伏频率值1/500;用HIV潜伏细胞测试dCas9基因激活系统激活HIV潜伏细胞频率1/615与对照SAHA激活剂激活HIV潜伏细胞频率1/749相比,更接近于设定频数1/500。结论 dCas9基因激活系统改进HIV体外扩增,可以提高HIV体外扩增检测的效率和灵敏度。
Objective To establish an enhanced HIV outgrowth assay approach combining a dcas9 activation model, improving test sensitivity by the induced activation of HIV sequence for amplified viral load reading and detection.Methods 1) 20 gRNAs sequences were constructed with Broad Institute gRNA Designer, targeting the LTR sequence of HIV-DNA. 2) luciferase reporters were fused to the HIV sequence and transfected to a Jurkat cell line with a dCas9 activation system. The screening of gRNAs was performed based on luciferase intensity, acquiring the optimal gRNA design. The optimal gRNA-L along with the dCase9 activation model were transfected into Jurkat cells, establish working cell lines Jurkat6465-L with efficient HIV amplification. 3)Wild-type HIV-luciferase infected Jurkat cells were cultured for the replication of HIV latency(Latent pre-set frequency is 1∶500). These cells were separately cultured on the same well dish with the working cell line cells. Luciferase intensity was observed and measured to characterize the detection capability of the amplification model on 7 d and 14 d. 4)A chemical-based HIV activation reagent(SAHA) was adopted to further evaluate the performance of the dCas9 amplification model.Results Acquired two optimal gRNA designs named gRNA-L and gRNA-O. Compared with the luciferase intensity of the blank controls [(9 876±437)pg/mL]on 6 d, significant amplification of HIV-Luc was observed at(85 530±979.2)pg/mL and(96 925±2 759)pg/mL(P<0.01). gRNA-L was preferred over the other one for superior performance. The namely Jurkat 6465 L cell line was constructed by transfecting gRNA-L and the dCas9 activation protein complex as the working cells for HIV latency analysis. On 7 and 14 d, The frequency of activating HIV latent model cells was 1/864 and 1/755 which were closer to the pre-set frequency(1∶500). The dCas9 activation model was further evaluated in comparison with a chemical-based HIV induction reagent SAHA. The dCas9 model yielded results with frequency readings(1∶615)closer to the pre-set frequency(1∶500) than the regent-based control(1∶749).Conclusion The dCas9 activation model improves the HIV outgrowth assay with proper accuracy and efficiency.
Objective To establish an enhanced HIV outgrowth assay approach combining a dcas9 activation model, improving test sensitivity by the induced activation of HIV sequence for amplified viral load reading and detection.Methods 1) 20 gRNAs sequences were constructed with Broad Institute gRNA Designer, targeting the LTR sequence of HIV-DNA. 2) luciferase reporters were fused to the HIV sequence and transfected to a Jurkat cell line with a dCas9 activation system. The screening of gRNAs was performed based on luciferase intensity, acquiring the optimal gRNA design. The optimal gRNA-L along with the dCase9 activation model were transfected into Jurkat cells, establish working cell lines Jurkat6465-L with efficient HIV amplification. 3)Wild-type HIV-luciferase infected Jurkat cells were cultured for the replication of HIV latency(Latent pre-set frequency is 1∶500). These cells were separately cultured on the same well dish with the working cell line cells. Luciferase intensity was observed and measured to characterize the detection capability of the amplification model on 7 d and 14 d. 4)A chemical-based HIV activation reagent(SAHA) was adopted to further evaluate the performance of the dCas9 amplification model.Results Acquired two optimal gRNA designs named gRNA-L and gRNA-O. Compared with the luciferase intensity of the blank controls [(9 876±437)pg/mL]on 6 d, significant amplification of HIV-Luc was observed at(85 530±979.2)pg/mL and(96 925±2 759)pg/mL(P<0.01). gRNA-L was preferred over the other one for superior performance. The namely Jurkat 6465 L cell line was constructed by transfecting gRNA-L and the dCas9 activation protein complex as the working cells for HIV latency analysis. On 7 and 14 d, The frequency of activating HIV latent model cells was 1/864 and 1/755 which were closer to the pre-set frequency(1∶500). The dCas9 activation model was further evaluated in comparison with a chemical-based HIV induction reagent SAHA. The dCas9 model yielded results with frequency readings(1∶615)closer to the pre-set frequency(1∶500) than the regent-based control(1∶749).Conclusion The dCas9 activation model improves the HIV outgrowth assay with proper accuracy and efficiency.
引文
[1] 中国疾病预防控制中心.中国艾滋病诊疗指南(2018版).协和医学杂志,2019,10(1):31-52.
[2] TW Chun,Lieven Stuyver,Stephanie B.Mizell,et al.Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy.Proc Natl Acad Sci,1997,94(24):13193-13200.
[3] Finzi D,Hermankova M,Pierson T,et al.Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy.Science,1997,278(5341):1295-1300.
[4] Wong JK,Hezareh M,Huldrych F,et al.Recovery of replication-competent HIV despite prolonged suppression of plasma viremia.Science,1997,278(5341):1291-1295.
[5] Siliciano JD,Siliciano RF.Enhanced culture assay for detection and quantitation of latently infected,resting CD4+ T-cells carrying replication-competent virus in HIV-1-infected individuals.Methods Mol Biol,2005,304(1):3-15.
[6] Ho YC,Shan L,Hosmane NN,Wang J,et al.Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure.Cell,2013,155(3):540-551.
[7] Konermann S,Brigham MD,Trevino AE,et al.Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.Nature,2015,517(7536):583-588.
[8] Zhang Y,Yin C,Zhang T,et al.CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific,persistent and robust reactivation of the HIV-1 latent reservoirs.Sci Rep,2015,5(16):277-288.
[9] Barczak W,Suchorska W,Rubis' B,et al.Universal real-time PCR-based assay for lentiviral titration.Mol Biotechnol.2015,57(2):195-200.
[10] Van Praag,Prins JM,Roos MTL,et al.OKT3 and IL-2 treatment for purging of the latent HIV-1 reservoir in vivo results in selective long-lasting CD4+ T cell depletion.J Clin Immunol,2001,21(3):218-26.
[11] Byrareddy SN,Arthos J,Cicala,et al.Sustained virologic control in SIV+ macaques after antiretroviral and alpha4 beta7 antibody therapy.Science,2016,354(6309):197-202.
[12] Fun A,Mok HP,Wills MR,et al.A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir.Sci Rep,2017,24(7):43231.
[13] Laird GM,Eisele EE,Rabi SA,et al.Rapid quantification of the latent reservoir for HIV-1 using a viral outgrowth assay.PLoS Pathog,2013,9(5):e1003398.
[14] Procopio FA,Fromentin R,Kulpa DA,et al.A novel assay to measure the magnitude of the inducible viral reservoir in HIV-infected individuals.E Bio Med,2015,2(8):874-883.
[15] Sanyal A,Mailliard RB,Rinaldo CR,et al.Novel assay reveals a large,inducible,replication-competent HIV-1 reservoir in resting CD4+ T cells.Nat Med,2017,23(7):885-889.
[2] TW Chun,Lieven Stuyver,Stephanie B.Mizell,et al.Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy.Proc Natl Acad Sci,1997,94(24):13193-13200.
[3] Finzi D,Hermankova M,Pierson T,et al.Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy.Science,1997,278(5341):1295-1300.
[4] Wong JK,Hezareh M,Huldrych F,et al.Recovery of replication-competent HIV despite prolonged suppression of plasma viremia.Science,1997,278(5341):1291-1295.
[5] Siliciano JD,Siliciano RF.Enhanced culture assay for detection and quantitation of latently infected,resting CD4+ T-cells carrying replication-competent virus in HIV-1-infected individuals.Methods Mol Biol,2005,304(1):3-15.
[6] Ho YC,Shan L,Hosmane NN,Wang J,et al.Replication-competent noninduced proviruses in the latent reservoir increase barrier to HIV-1 cure.Cell,2013,155(3):540-551.
[7] Konermann S,Brigham MD,Trevino AE,et al.Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.Nature,2015,517(7536):583-588.
[8] Zhang Y,Yin C,Zhang T,et al.CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific,persistent and robust reactivation of the HIV-1 latent reservoirs.Sci Rep,2015,5(16):277-288.
[9] Barczak W,Suchorska W,Rubis' B,et al.Universal real-time PCR-based assay for lentiviral titration.Mol Biotechnol.2015,57(2):195-200.
[10] Van Praag,Prins JM,Roos MTL,et al.OKT3 and IL-2 treatment for purging of the latent HIV-1 reservoir in vivo results in selective long-lasting CD4+ T cell depletion.J Clin Immunol,2001,21(3):218-26.
[11] Byrareddy SN,Arthos J,Cicala,et al.Sustained virologic control in SIV+ macaques after antiretroviral and alpha4 beta7 antibody therapy.Science,2016,354(6309):197-202.
[12] Fun A,Mok HP,Wills MR,et al.A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir.Sci Rep,2017,24(7):43231.
[13] Laird GM,Eisele EE,Rabi SA,et al.Rapid quantification of the latent reservoir for HIV-1 using a viral outgrowth assay.PLoS Pathog,2013,9(5):e1003398.
[14] Procopio FA,Fromentin R,Kulpa DA,et al.A novel assay to measure the magnitude of the inducible viral reservoir in HIV-infected individuals.E Bio Med,2015,2(8):874-883.
[15] Sanyal A,Mailliard RB,Rinaldo CR,et al.Novel assay reveals a large,inducible,replication-competent HIV-1 reservoir in resting CD4+ T cells.Nat Med,2017,23(7):885-889.