Abstract
HIV/AIDS remains a major health threat despite significant advances in the prevention and treatment of HIV infection. The major reason is the inability of existing treatments to eradicate the multiple HIV reservoirs in the human body, including astrocytes in the human brain. CRISPR/Cas9 system is an emerging gene-editing technique with the potential to eliminate or disrupt HIV provirus in HIV reservoir cells, which may lead to a complete cure of HIV/AIDS. The key components of CRISPR/Cas9 are guide RNAs (gRNAs) which determine specific sequence targeting of DNAs. This study established a novel, simple and quick screening method to identify gRNA candidates for targeting HIV provirus in astrocytes. Briefly, stable astrocytes clones with an integrated fluorescent HIV reporter and Cas9 expression gene were generated. Various gRNAs were screened for their efficiencies against HIV provirus in these cells. Moreover, these gRNAs and Cas9 protein were successfully tested on HIV latent astrocytes without Cas9 expression to mimic clinical conditions. HIV provirus gene-editing were confirmed by cell genomic DNA PCR and fluorescent marker expression analysis. In the future, the established transgenic cells can be used for other gene-editing studies and is well-suited for high-throughput screen application.
Highlights
Human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) is a serious pandemic disease that remains a major threat to human health
There are thousands of guide RNAs (gRNAs) could be designed against 10 kb HIV provirus sequence, a screen tool is in high demand for the selection of effective and high impact gRNA for HIV provirus disruption
NeuroAIDS is a severe threat for HIV/AIDS patients and the major barrier for treating NeuroAIDs is the HIV latent cells in patients’ brains, including astrocytes, microglial cells and macrophages
Summary
Human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) is a serious pandemic disease that remains a major threat to human health. The breakthrough of gene-editing technology raises the hope for eradication of HIV because the provirus may be efficiently manipulated in the host cell genome through the newly developed technique of CRISPR/Cas[9] system. In HIV gene therapy field, there are no simple and quick screening methods for the identification of strong gRNA candidates for the removal or disruption of HIV provirus from HIV reservoir cells. Molecular and cell biology approaches were applied to establish an in vitro screen platform based on a newly created transgenic astrocyte with a HIV reporter and Cas[9] expression gene. As far as we know, this paper is the first to apply a bioengineering approach to establish a brain cell line gRNA screen platform This platform can be very helpful for researchers to screen and test the efficiencies of gRNA candidates for their gene-editing treatment of HIV latent cells
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