%0 Journal Article %A Li, Chang %A Guan, Xinmeng %A Du, Tao %A Jin, Wei %A Wu, Biao %A Liu, Yalan %A Wang, Ping %A Hu, Bodan %A Griffin, George E. %A Shattock, Robin J. %A Hu, Qinxue %T Inhibition of HIV-1 infection of primary CD4+ T-cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9 %D 2015 %J Journal of General Virology, %V 96 %N 8 %P 2381-2393 %@ 1465-2099 %R https://doi.org/10.1099/vir.0.000139 %I Microbiology Society, %X CCR5 serves as an essential coreceptor for human immunodeficiency virus type 1 (HIV-1) entry, and individuals with a CCR5Δ32 variant appear to be healthy, making CCR5 an attractive target for control of HIV-1 infection. The CRISPR/Cas9, which functions as a naturally existing adaptive immune system in prokaryotes, has been recently harnessed as a novel nuclease system for genome editing in mammalian cells. Although CRISPR/Cas9 can be readily delivered into cell lines, due to the large size of the Cas9 protein, efficient delivery of CCR5-targeting CRISPR/Cas9 components into primary cells, including CD4+ T-cells, the primary target for HIV-1 infection in vivo, remains a challenge. In the current study, following design of a panel of top-ranked single-guided RNAs (sgRNAs) targeting the ORF of CCR5, we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of the CCR5 locus in cell lines, resulting in the knockout of CCR5 expression on the cell surface. Next-generation sequencing revealed that various mutations were introduced around the predicted cleavage site of CCR5. For each of the three most effective sgRNAs that we analysed, no significant off-target effects were detected at the 15 top-scoring potential sites. More importantly, by constructing chimeric Ad5F35 adenoviruses carrying CRISPR/Cas9 components, we efficiently transduced primary CD4+ T-lymphocytes and disrupted CCR5 expression, and the positively transduced cells were conferred with HIV-1 resistance. To our knowledge, this is the first study establishing HIV-1 resistance in primary CD4+ T-cells utilizing adenovirus-delivered CRISPR/Cas9. %U https://www.microbiologyresearch.org/content/journal/jgv/10.1099/vir.0.000139