PDF(Pubmed)
Abstract：
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing system has been shown to be effective at inhibiting human immunodeficiency virus type 1 (HIV-1). Studies have not consistently used a trackable dual reporter system to determine what cells received the Cas9/gRNA to determine the overall knockdown of HIV. Some studies have used stably transduced cells under drug selection to accomplish this goal. Here a two-color system was used that allows tracking of viral protein expression and which cells received the CRISPR/Cas9 system. These experiments ensured that each gRNA used was a perfect match to the intended target to remove this variable. The data showed that gRNAs targeting the transactivation response element (TAR) region or other highly conserved regions of the HIV-1 genome were effective at stopping viral gene expression, with multiple assays demonstrating greater than 95 percent reduction. Conversely, gRNAs targeting conserved sites of the 5\' portion of the U3 region were largely ineffective, demonstrating that the location of edits in the long terminal repeat (LTR) matter with respect to function. In addition, it was observed that a gRNA targeting Tat was effective in a T-cell model of HIV-1 latency. Taken together, these studies demonstrated gRNAs designed to highly conserved functional regions have near 100% efficacy in vitro in cells known to have received the Cas9/gRNA pair.
摘要：
聚集的定期间隔短回文重复（CRISPR）/Cas9基因编辑系统已被证明可有效抑制人类免疫缺陷病毒1型（HIV-1）。研究并没有一致地使用可追踪的双报告系统来确定哪些细胞接受了Cas9/gRNA来确定HIV的整体敲除。一些研究已经在药物选择下使用稳定转导的细胞来实现该目标。这里使用双色系统,其允许追踪病毒蛋白表达和哪些细胞接受CRISPR/Cas9系统。这些实验确保所使用的每个gRNA与预期靶标完美匹配以去除该变量。数据显示，针对HIV-1基因组的反式激活反应元件（TAR）区域或其他高度保守区域的gRNA有效地阻止病毒基因表达，多项检测显示减少了95%以上。相反，靶向U3区5'部分保守位点的gRNAs在很大程度上是无效的，证明长末端重复(LTR)中编辑的位置与功能有关。此外,观察到靶向Tat的gRNA在HIV-1潜伏期的T细胞模型中是有效的。一起来看,这些研究表明,在已知接受Cas9/gRNA对的细胞中,设计成高度保守功能区域的gRNA具有接近100%的体外功效.
