1887

Abstract

CCR5 serves as an essential coreceptor for human immunodeficiency virus type 1 (HIV-1) entry, and individuals with a CCR5 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 , remains a challenge. In the current study, following design of a panel of top-ranked single-guided RNAs (sgRNAs) targeting the ORF of , we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of the 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 . 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.

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2015-08-01
2019-10-22
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