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 SpCas9-127_HBX2) 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.

中文翻译：

---

分析 CRISPR/Cas9 Guide RNA 针对遗传多样性 HIV-1 分离株的效率和特异性。


使用 CRISPR/Cas9 的基因编辑方法正在开发中，作为针对整合的 HIV-1 原病毒的一种手段。由于对这种方法的特异性和有效性的担忧，人们对使用基因编辑作为抗 HIV-1 疗法的热情有所减弱。针对多种遗传多样性 HIV-1 分离株中保守序列的引导 RNA (gRNA) 将具有更大的临床实用性。然而，应在脱靶裂解事件的背景下考虑中靶功效，因为这些可能构成基于 CRISPR 的治疗的基本安全参数。我们分析了一组针对 HIV-1 5' 和 3' 长末端重复 (LTR) 区域的化脓性链球菌Cas9 ( Sp Cas9) gRNA。我们使用具有遗传多样性的 HIV-1 LTR 序列的体外裂解测定来确定跨 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 前病毒序列的靶向性（体内）。在体外和体内实验中，我们观察到通过同时使用两种不同的 gRNA 可以提高序列破坏的效率。接下来，利用 CIRCLE-Seq 使用来自整合了 HIV-1 前病毒 DNA 的细胞的基因组 DNA 来识别脱靶切割事件。我们鉴定了一种靶向 LTR U3 区域的 gRNA（称为Sp Cas9-127 _HBX2 ），对来自遗传多样性 HIV-1 毒株的序列具有广泛的切割效率。基于这些结果，我们提出了识别和开发抗 HIV CRISPR 疗法的工作流程。