Use CRISPR-Cas Gene Editing Technology To Design Targeted HIV-1 Genome To Inhibit Virus Replication Research

Acquired immunodeficiency syndrome(AIDS)caused by human immunodeficiency virus(HIV)infection still remains a major global public health problem.Although antiretroviral therapy(ART)is effective in suppressing viral replication and reducing viral load in HIV patients,it does not offer a permanent cure.The ultimate cure for HIV/AIDS will be the removal or disruption of integrated HIV provirus from latently infected cells or the elimination of these latent cells completely.The breakthrough of gene-editing technology raises the hope for eradication of HIV as the provirus can be removed from the host cell genome.The clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)system is an emerging gene-editing technique with the potential to eliminate or disrupt HIV-integrated genomes or HIV-infected cells from HIV reservoirs,inhibit viral infection and replicaton,which could result in the complete cure of HIV/AIDS.Casl3a is a RNA-guided RNase which requires the activity of its two HEPN domains to facilitate ssRNA cleavage.At present,researches about CRISPR/Cas9 inhibiting HIV infection are mianly foucused on editing the integrated HIV proviral genome.After cleavage,the double strand DNA break is often repaired by the non-homologous end joining(NHEJ)machinery,resulting in deletions or insertions(indels).Except the integrated HIV-1 DNA,dose the CRISPR/Cas9 target HIV-1 DNA before integration?Which steps of HIV-1 life cycle could be targeted by CRISPR/Cas9?In additon,it remains to be studied whether HIV-1 replication could be inhibited by CRISPR/Cas13a targeting HIV-1 RNA,which is a very important research direction by interfering HIV replication or eliminating HIV-infected cells.In this study,we have tested the gene editing system in inhibiting HIV-1 infection by targeting the viral genome.We observed strong inhibition of HIV-1 infection by Cas9/sgRNA.Sequencing analysis indicated that each target site was efficiently mutated upon Cas9/sgRNA treatment,with indels prevailing.Further,real-time PCR was performed to determine the levels of newly synthesized HIV-1 DNA,including the early DNA,late DNA and the integrated DNA products.Results showed that Cas9 leads to reduction in the levels of viral cDNA products including the late reverse transcription virus DNA and integrated viral DNA.This defect might have reflected the degradation of viral DNA that has not been immediately repaired after Cas9 cleavage.Since HIV-1 reverse transcription takes place in the cytoplasm,we further tested whether Cas9 is also able to cleave the newly synthesized HIV-1 DNA in the cytoplasm and thus inhibit HIV-1 infection.We utilized a Cas9 without NLS signal(Cas9-delNLS),which was strictly located in the cytoplasm.Results showed that Cas9-delNLS/gRNA inhibited infected HIV-1 gene expression to a similar extent as Cas9-NLS/gRNA did,but the Cas9-delNLS/gRNA is unable to edit and excise the integrated HIV-1 DNA.Moreover,in addition to targeting HIV DNA,this study also applied CRISPR/Cas13a system to target HIV RNA and inhibit HIV replication in mammalian cell.First,the efficiency of CRISPR/Cas13a editing was tested by targeting GFP RNA in 293T.Then,crRNAs targeting HIV-1 LTR R region,tat,rev and gag RNA were designed according to the design principles of LbuCasl3a and the function of HIV-1 gene.The results showed that HIV-1 replication was significantly interfered by CRISPR/Cas 13a targeting LTR promotor or gene coding region,and the interference depended on the RNase activity of Cas13a.Collectively,in this study,we comprehensively studied the role of CRISPR/Cas9 in the HIV life cycle.Results suggest that Cas9/sgRNA is able to target and edit HIV-1 DNA both in the cytoplasm and in the nucleus.The inhibitory effect of Cas9 on HIV-1 is attributed to both the indels in viral DNA and the reduction in the levels of viral DNA.Meanwhile,targeting the HIV-1 RNA,CRISPR/Cas13a inhibited HIV-1 replication.It is expected to combine Cas9 and Casl3a to cross-edit HIV-1 DNA and RNA.These findings provide an important reference for the prevention and treatment of HIV-1 infection.