Suppressing HIV infection by disrupting CCR5 with double nicking CRISPR-Cas9 syst

产品说明：使用基因组工程工具锌指核酸酶（ZFN）破坏HIV感染患者CD 4 + T细胞中的CCR 5基因的细胞治疗临床研究的最新结果显示了令人印象深刻的结果。II型原核CRISPR-Cas9系统是一类用于靶向基因组工程的新工具。最近的报道显示，细菌内切核酸酶Cas9与小向导RNA（gRNA）的组合可以用于编辑基因组序列，其效率与ZFN和TALEN相当或甚至更高。通过测试不同版本的Cas9酶和gRNA靶向CCR 5基因，我们确定了最佳的gRNA和Cas9组合，其CCR 5破坏效率显着高于全面优化的ZFN。不幸的是，最近的研究表明，CRISPR系统介导的基因组编辑通常具有广泛的脱靶效应，使其不适合治疗应用。然而，最近的另一项研究表明，将Cas9切口酶突变体（D10 A）与成对的gRNA组合以在靶基因组位点产生双切口可以显著降低脱靶效应。我们已经鉴定了两对靶向CCR 5的gRNA，其与Cas9（D10 A）组合，有效地敲除CCR 5。在这里，我们提出在Hu-PBL小鼠模型中评估双切口CRISPR-Cas9系统的抗HIV功效和脱靶效应。该项目的长期目标是开发一种ZFN的替代HIV疗法，可能为HIV患者提供具有良好安全性的功能性治疗。

DESCRIPTION: Recent results from clinical studies of cellular therapy using the genome-engineering tool zinc finger nuclease (ZFN) to disrupt the CCR5 gene in CD4+ T cells from HIV-infected patients have shown impressive results. The Type II prokaryotic CRISPR-Cas9 system is a new class of tool for targeted genome engineering. Recent reports showed that the bacterial endonuclease Cas9 in combination with a small guide RNA (gRNA) can be used to edit genome sequence with an efficiency comparable to or even greater than ZFN and TALEN. By testing different versions of the Cas9 enzyme and gRNA in targeting the CCR5 gene, we have identified the optimal gRNA and Cas9 combination, which has significantly higher CCR5-disruption efficiency than comprehensively optimized ZFN. Unfortunately, recent studies have shown that CRISPR system-mediated genome editing generally has extensive off- target effects, making it unsuitable for therapeutic applications. However, another recent study showed that combining a Cas9 nickase mutant (D10A) with paired gRNAs to create double nicking at the target genome site can dramatically lower off-target effects. We have identified two pairs of gRNAs targeting CCR5 that, in combination with Cas9 (D10A), efficiently knock out CCR5. Here we propose to evaluate the anti-HIV efficacy and off-target effects of the double-nicking CRISPR-Cas9 system in the Hu-PBL mouse model. The long-term goal of this project is to develop an alternative HIV therapy to ZFN that might offer a functional cure with excellent safety profile for HIV patients.