Inhibition of HIV-1 infection of primary CD4+ T cells by gene editing of CCR5 using adenovirus-delivered CRISPR/Cas9.

　　Li C, Guan X, Du T, Jin W, Wu B, Liu Y, Wang P, Hu B, Griffin GE3, Shattock RJ, Hu Q.

      J Gen Virol. 2015 Apr 8. pii: vir.0.000139. doi: 10.1099/vir.0.000139.

　　Abstract 

　　CCR5 serves as an essential coreceptor for HIV-1 entry and individuals with a CCR5Δ32 variant appears to be healthy, making CCR5 an attractive target for control of HIV-1 infection. The CRISPR/Cas9, that functions as a naturally-existed 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 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 RNA (sgRNA) targeting the open reading frame of CCR5, we demonstrate that CRISPR/Cas9 can efficiently mediate the editing of CCR5 locus in cell lines, resulting in the knockout of CCR5 expression on 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 analyzed, no significant off-target effects were detected at the 15 top-scored 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.

http://www.ncbi.nlm.nih.gov/pubmed/25854553