Abstract
Viral DNA integrated in host cells is a major barrier to completely curing HIV-1. However, genome editing using the recently developed technique of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has the potential to eradicate HIV-1. The present study aimed to use a lentiviral vector-based CRISPR/Cas9 system combined with dual-small/single guide RNAs (sgRNAs) to attack HIV-1 DNA in the latency reactivation model J-Lat 10.6 cell line and to assess off-target effects using whole-genome sequencing (WGS). We designed 12 sgRNAs targeting HIV-1 DNA, and selected high-efficiency sgRNAs for further pairwise combinations after a preliminary evaluation of the editing efficiency. Three combinations of dual-sgRNAs/Cas9 with high editing efficiency were screened successfully from multiple combinations. Among these combinations, the incidences of insertions and deletions in the sgRNA-targeted regions reached 76% and above, and no credible off-target sites were detected using WGS. The results provided comprehensive basic experimental evidence and methodological recommendations for future personalized HIV-1 treatment using CRISPR/Cas9 genome editing technology.
Highlights
By the end of 2019, worldwide, 38 million people were estimated to be living with HIV
Our study focused on attacking HIV-1 DNA using clustered regularly interspaced short palindromic repeats (CRISPR) therapy, for which we selected the latency reactivation model
Based on the latent HIV-1 genome sequence of the J-Lat 10.6 cell line, a total of 12 single guide RNAs (sgRNAs) with a high on-target score, which targeted different protein-coding sequences, such as gag, pol, tat, and env genes, respectively, were designed using the online CRISPR design tool (Hsu et al, 2013; Figure 1; Table 1). Of these 12 target sites, five sgRNAs were located in the gag gene, four sgRNAs were in the pol gene, one sgRNA was in the tat gene, and two were in the env gene
Summary
By the end of 2019, worldwide, 38 million people were estimated to be living with HIV. According to a WHO report, there were still about 1.7 million new HIV infections in 2019, and the HIV epidemic poses a major threat to global human health. The majority of patients with HIV/AIDS treated with these antiviral drugs were able to achieve the virological inhibition criterion that the viral load in plasma was below the detection limit (Laskey and Siliciano, 2014). Current antiviral therapy cannot completely remove the virus from the human body because of the stable integration of proviral DNA into the cellular genome. Genome editing technology might be one of the solutions to remove proviral DNA from HIV-1 infected cells
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