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


 CONCLUSIONS
 Our study provides a basis for possible application of CXCR4-targeted genome editing by CRISPR/SaCas9 in HIV-1 gene therapy.
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