The first use of the CRISPR/Cas9 system to disrupt HIV-1 proviral DNA was reported in 2013. These results have since been validated in vitro and in a rodent model. Enthusiasm for CRISPR/Cas9-based HIV-1 eradication approaches arises in part from sequence specificity and, in contrast with so-called “shock-and-kill” treatments, potential avoidance of cell activation and toxicity. However, off-target activity of Cas9 has been reported. There are varying estimates of the prevalence of off-target effects, and attempts have been made to minimize these effects through genetic engineering of Cas proteins. Nevertheless, in the HIV-1 patient, the gene editing system would have to be introduced into a large number of cells to reduce the size of known HIV-1 reservoirs; even a vanishingly small percentage of off-target activity could result in potentially dangerous genomic alterations. Viral evolution in response to Cas9 has also raised some concerns.
An alternative application of CRISPR/Cas9 in HIV-1 studies seeks to achieve the opposite goal: upregulation of HIV expression instead of disruption or excision. In this approach, a nuclease-mutated Cas9 (dead Cas9 or dCas9) fused to a transcriptional activator domain reverses HIV-1 latency. Instead of cutting genomic DNA, the dCas9 transcriptional activator stimulates HIV-1 transcription, resulting in increased display of viral products and possible elimination of HIV-1 harboring cells by the immune system. The feasibility of this approach has been demonstrated in vitro by several groups.