使用CRISPR/Cas9的基因编辑方法正在被开发为靶向整合的HIV-1前病毒的手段。对使用基因编辑作为抗HIV-1治疗剂的热情已被对这种方法的特异性和功效的担忧所削弱。靶向广泛的遗传多样性HIV-1分离物的保守序列的引导RNA(gRNA)将具有更大的临床效用。然而,应在脱靶裂解事件的背景下考虑中靶功效,因为这些事件可能包含基于CRISPR的治疗剂的基本安全性参数.我们分析了一组针对HIV-1的5'和3'长末端重复(LTR)区域的化脓性链球菌Cas9(SpCas9)gRNA。我们使用具有遗传上不同的HIV-1LTR序列的体外切割测定法来确定跨HIV-1进化枝的gRNA活性。gRNA/Cas9核糖核蛋白的基于脂质的转染用于评估细胞中整合的HIV-1前病毒序列的靶向(体内)。对于体外和体内实验,通过同时使用两种不同的gRNA,我们观察到序列破坏的效率提高.接下来,CIRCLE-Seq用于使用来自具有整合的HIV-1前病毒DNA的细胞的基因组DNA鉴定脱靶切割事件。我们鉴定了靶向LTR的U3区域的gRNA(称为SpCas9-127HBX2),其具有针对来自遗传上不同的HIV-1毒株的序列的宽切割效率。基于这些结果,我们提出了鉴定和开发抗HIVCRISPR疗法的工作流程。
Gene editing approaches using CRISPR/Cas9 are being developed as a means for targeting the integrated HIV-1 provirus. Enthusiasm for the use of gene editing as an anti-HIV-1 therapeutic has been tempered by concerns about the specificity and efficacy of this approach. Guide RNAs (gRNAs) that target conserved sequences across a wide range of genetically diverse HIV-1 isolates will have greater clinical utility. However, on-target efficacy should be considered in the context of off-target cleavage events as these may comprise an essential safety parameter for CRISPR-based therapeutics. We analyzed a panel of Streptococcus pyogenes Cas9 (SpCas9) gRNAs directed to the 5\' and 3\' long terminal repeat (LTR) regions of HIV-1. We used in vitro cleavage assays with genetically diverse HIV-1 LTR sequences to determine gRNA activity across HIV-1 clades. Lipid-based transfection of gRNA/Cas9 ribonucleoproteins was used to assess targeting of the integrated HIV-1 proviral sequence in cells (in vivo). For both the in vitro and in vivo experiments, we observed increased efficiency of sequence disruption through the simultaneous use of two distinct gRNAs. Next, CIRCLE-Seq was utilized to identify off-target cleavage events using genomic DNA from cells with integrated HIV-1 proviral DNA. We identified a gRNA targeting the U3 region of the LTR (termed SpCas9-127HBX2) with broad cleavage efficiency against sequences from genetically diverse HIV-1 strains. Based on these results, we propose a workflow for identification and development of anti-HIV CRISPR therapeutics.