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.