Potent and targeted activation of HIV-1 using the CRISPR/Cas9 activator Complex

Integration of the HIV-1 provirus in the host genome ensures a persistent supply of latently infected cells. This latent reservoir is recalcitrant to antiretroviral therapy (ART) making lifelong treatment the only option for patients. The “shock and kill” strategy aims to eradicate latent HIV by reactivating proviral gene expression followed by ART treatment. Gene specific transcriptional activation can be achieved using the RNA-guided CRISPR-Cas9 system comprising small guide RNAs (sgRNAs) with a nuclease deficient Cas9 mutant (dCas9) fused to the VP64 transactivation domain (dCas9-VP64).  We engineered this system to target 23 sites within the LTR promoter of HIV-1 and identified a “hotspot” for activation. We studied the functionality of activating sgRNAs to transcriptionally modulate the latent proviral genome across multiple different in vitro latency cell models including several J-Lat, ACH2 J1.1 and the CEM T cell model comprising a single clonal population of integrated mCherry-IRES-Tat from a full-length HIV LTR (LChIT).  While we observed variable responses of latent cell models to well-characterized chemical stimuli, we detected consistent efficient activation of latent virus mediated by activator sgRNAs.  In addition, transcriptome analysis of chemically treated cells revealed massive non-specific gene dysregulation whereas by comparison, dCas9-VP64/sgRNAs induced specific activation of the integrated provirus.  In conclusion, we show the potential for CRISPR-mediated gene activation systems to provide enhanced efficiency and specificity in a targeted latency reactivation strategy. This represents a promising approach to a “functional cure” of HIV/AIDS. Overall design: Three experimental conditions (sgRNA control, TNF treated and sgRNA against the LTR of HIV-1) were analyzed in triplicate using two sequencing lanes

HIV-1前病毒整合至宿主基因组后，可持续产生潜伏感染细胞。该潜伏库对抗逆转录病毒治疗（antiretroviral therapy, ART）耐受，致使患者需终身接受治疗。“激活并清除（shock and kill）”策略旨在通过重新激活前病毒基因表达，联合ART治疗以根除潜伏HIV。利用搭载融合了VP64反式激活结构域（VP64 transactivation domain）的核酸酶缺陷型Cas9突变体（nuclease deficient Cas9 mutant, dCas9）与单引导RNA（small guide RNAs, sgRNAs）的RNA引导CRISPR-Cas9系统，可实现基因特异性转录激活。本研究对该系统进行工程化改造，靶向HIV-1长末端重复序列（long terminal repeat, LTR）启动子内的23个位点，并鉴定出一个转录激活热点区域。我们针对激活型sgRNAs的功能展开研究，以在多种体外（in vitro）潜伏细胞模型中对潜伏前病毒基因组进行转录调控，所涉模型包括数种J-Lat、ACH2 J1.1细胞系，以及由全长HIV LTR驱动整合型mCherry-IRES-Tat的单克隆CEM T细胞模型（LChIT）。尽管我们观察到不同潜伏细胞模型对已明确表征的化学刺激响应存在差异，但检测到激活型sgRNAs介导的潜伏病毒高效且一致的激活效果。此外，对经化学试剂处理细胞的转录组分析（transcriptome analysis）显示，其存在广泛的非特异性基因表达失调（gene dysregulation）；相较之下，dCas9-VP64/sgRNAs仅特异性激活整合型前病毒。综上，本研究证实CRISPR介导的基因激活系统可在靶向性潜伏激活策略中提升效率与特异性，该方法有望为HIV/AIDS实现“功能性治愈（functional cure）”提供新途径。实验整体设计：设置三组实验条件（sgRNA对照组、TNF处理组、靶向HIV-1 LTR的sgRNA组），每组设置三次生物学重复，采用两条测序泳道完成测序。