In vitro validation of CRISPR-Cas9 mediated targeting of transgene integration sites to excise HIV-1 sequences from HIV-1 Tg rat

Persistent expression of HIV-1 proteins in the absence of active viral replication in HIV-1 Tg rat model resembles pathologies observed in ART-controlled HIV-1 positive patients. HIV-1 transgene expression in different tissues leads to progressive HIV-1 proteinotoxicity, resulting in metabolic and behavioral malfunctions, which can be further intensified by the use of addictive substances. Recently whole genome sequence analysis was performed leading to identification of the sites of HIV-1 transgene integration in the HIV-1 Tg rat genome. Using this information, we developed a PCR based genotyping and qPCR techniques to accurately identify and quantify transgene positive cells/tissues. The two HIV-1 transgene integration sites in chromosome 10 and 13 were confirmed by Sanger sequencing of PCR amplicons spanning rat and HIV-1 sequences. Based on these data we were able to develop a new, simplified CRISPR-Cas9 mediated transgene excision strategy. Two pairs of gRNAs each targeting 5’ and 3’ flanking regions of integration sites in chromosome 10 and 13 were designed and cloned into AAV and lentiviral delivery vectors. Next, the constructs were tested in vitro using primary rat embryo fibroblasts derived from the single litter of HIV-1 Tg rats and rat neuronal cell line. CRISPR-Cas9 mediated excision of transgene sequences resulted in inhibition of HIV-1 expression in treated cells. Successful removal of transgene sequences from different tissues and organs of transgenic animals should limit and/or reverse HIV-1 protein expression mediated pathologies.