Excision of HIV-1 DNA by gene editing: a proof-of-concept in vivo study

Gene Ther. 2016 Aug;23(8-9):690-5. doi: 10.1038/gt.2016.41. Epub 2016 May 19.


A CRISPR/Cas9 gene editing strategy has been remarkable in excising segments of integrated HIV-1 DNA sequences from the genome of latently infected human cell lines and by introducing InDel mutations, suppressing HIV-1 replication in patient-derived CD4+ T-cells, ex vivo. Here, we employed a short version of the Cas9 endonuclease, saCas9, together with a multiplex of guide RNAs (gRNAs) for targeting the viral DNA sequences within the 5'-LTR and the Gag gene for removing critically important segments of the viral DNA in transgenic mice and rats encompassing the HIV-1 genome. Tail-vein injection of transgenic mice with a recombinant Adeno-associated virus 9 (rAAV9) vector expressing saCas9 and the gRNAs, rAAV:saCas9/gRNA, resulted in the cleavage of integrated HIV-1 DNA and excision of a 978 bp DNA fragment spanning between the LTR and Gag gene in the spleen, liver, heart, lung and kidney as well as in the circulating lymphocytes. Retro-orbital inoculation of rAAV9:saCas9/gRNA in transgenic rats eliminated a targeted segment of viral DNA and substantially decreased the level of viral gene expression in circulating blood lymphocytes. The results from the proof-of-concept studies, for the first time, demonstrate the in vivo eradication of HIV-1 DNA by CRISPR/Cas9 on delivery by an rAAV9 vector in a range of cells and tissues that harbor integrated copies of viral DNA.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • CD4-Positive T-Lymphocytes / metabolism
  • CRISPR-Cas Systems*
  • Cells, Cultured
  • DNA, Viral / genetics*
  • Dependovirus / genetics
  • Gene Editing / methods*
  • Gene Products, gag / genetics
  • Gene Targeting / methods
  • HIV-1 / genetics*
  • Kidney / metabolism
  • Liver / metabolism
  • Lung / metabolism
  • Mice
  • Myocardium / metabolism
  • Rats


  • DNA, Viral
  • Gene Products, gag