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.
Next-generation sequencing (NGS) is a trending new platform which allows cheap, quantitative, high-throughput, parallel sequencing for minority variants with frequencies less than 20% of the HIV-1 quasi-species. In clinical setting, these advantages are crucial for choosing antiretroviral drugs with low genetic barriers and will potentially benefit treatment outcomes.
In this investigation, we implemented the Boxin HIV-1 NGS platform for genotyping the drug-resistance-associated variants in PR/RT regions. Plasmids with known mutations were used to analyze the accuracy, reproducibility, and reliability of the Boxin NGS assay. Variant frequencies reported by Boxin NGS and the theoretical value were highly concordant. The Bland-Altman plot and the coefficient of variation (7%) suggested that the method has excellent reproducibility and reliability. Sanger sequencing confirmed the existence of these known variants with frequencies equal or above 20%.
78 blood samples were obtained from AIDS patients and underwent PR/RT region genotyping by Sanger sequencing and Boxin NGS. 33 additional drug resistance mutations were identified by Boxin NGS, 23/33 mutations were minority variants with frequencies below 20%.
15 blood samples obtained from AIDS patients underwent PR/RT region genotyping by Sanger sequencing, Boxin NGS, and Vela NGS. The Bland-Altman plot suggested that the variant frequencies detected by Boxin and Vela were highly concordant. Moreover, Boxin NGS assay detected five more minority variants with frequencies ranged from 1% to 20%. In a series of samples collected from 2016 to 2017, Boxin NGS reported a M184V mutation with a frequency of 4.92%, 3 months earlier than this mutation was firstly detected by Vela NGS and Sanger sequencing.
In conclusion, Boxin NGS had good accuracy, reproducibility, and reliability. Boxin NGS was highly concordant with Sanger sequencing and Vela NGS. In terms of genotyping HIV-1 variants in PR/RT regions, Boxin NGS was more cost-efficient and appeared to have increased sensitivity without compromising sequence accuracy.
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