Genome modification of CXCR4 by Staphylococcus aureus Cas9 renders cells resistance to HIV-1 infection

Qiankun Wang, Shuliang Chen, Qiaoqiao Xiao, Zhepeng Liu, Shuai Liu, Panpan Hou, Li Zhou, Wei Hou, Wenzhe Ho, Chunmei Li, Li Wu, Deyin Guo

The CRISPR/Cas9 system has been widely used for genome editing in mammalian cells. CXCR4 is a co-receptor for human immunodeficiency virus type 1 (HIV-1) entry, and loss of CXCR4 function can protect cells from CXCR4 (X4)-tropic HIV-1 infection, making CXCR4 an important target for HIV-1 gene therapy. However, the large size of the CRISPR/SpCas9 system presents an obstacle to its efficient delivery into primary CD4(+) T cells. Recently, a small Staphylococcus aureus Cas9 (SaCas9) has been developed as a genome editing tool can address this question. Therefore, it provides a promising strategy for HIV-1 gene therapy if it is used to target CXCR4. Here, we employed a short version of Cas9 from Staphylococcus aureus (SaCas9) for targeting CXCR4. We demonstrated that transduction of lenti-virus expressing SaCas9 and selected single-guided RNAs of CXCR4 in human CD4(+) T cell lines efficiently induced the editing of the CXCR4 gene, making these cell lines resistant to X4-tropic HIV-1 infection. Moreover, we efficiently transduced primary human CD4(+) T cells using adeno-associated virus-delivered CRISPR/SaCas9 and disrupted CXCR4 expression. We also showed that CXCR4-edited primary CD4(+) T cells proliferated normally and were resistant to HIV-1 infection. Our study provides a basis for possible application of CXCR4-targeted genome editing by CRISPR/SaCas9 in HIV-1 gene therapy.