| The human immunodeficiency virus type 1(HIV-1)epidemic still remains a major threat to global human health.Antiretroviral therapy(ART)can effectively control the acquired immunodeficiency syndrome(AIDS)disease progression and extend the life of HIV/AIDS patients.However,with the scale-up of antiretroviral therapy(ART)globally,some degree of HIV-1 drug resistance is anticipated.The emergence of HIV-1 drugresistant strains will cause the current antiretroviral drugs to face the risk of partial or complete invalidation,which would lead to the failure of virological treatment.It is particularly important to monitor HIV-1 drug resistance in time.On the other hand,a variety of antiretroviral drugs can exert antiviral effects on different stages of the HIV-1life cycle,but they cannot eradicate latent viral reservoirs.The third-generation genome editing technology CRISPR/Cas9 system has the potential to cure HIV-1 infection.Therefore,the following three parts of the research were conducted in this paper.In the first part,to understand the current subtype distribution and transmitted drug resistance level in Zhejiang province,we performed genotypic drug resistance testing of232 treatment-naive HIV-infected individuals recruited from 2014 to 2019.A total of 205protease(PR)+ reverse transcriptase(RT)genes and 52 Integrase(IN)genes were successfully amplified.The phylogenetic analysis indicated that the distribution of HIV-1 genotypes was dominantly CRF07＿BC(42.0%,86/205),CRF01＿AE(39.5%,81/205),B/B’ subtype(7.3%,15/205)and CRF08＿BC(6.3%,13/205),and many other subtypes co-existed.Drug resistance analysis demonstrated that the current transmitted drug resistance level in this region was 15.1%.The proportion of major protease inhibitors(PI)resistance mutations was 3.2%(7/205);major nucleoside reverse transcriptase inhibitors(NRTI)and non-nucleoside reverse transcriptase inhibitors(NNRTI)resistance mutations accounted for 8.8%(18/205)and 8.8%(18/205),respectively;the prevalence of major integrase strand transfer inhibitors(INSTI)resistance mutations was 1.9%(1/52).The prevalence of HIV-1 transmitted drug resistance in Zhejiang province was at a high level,especially resistance to NRTIs and NNRTIs.Therefore,it is necessary for treatment-naive HIV-infected individuals to perform HIV-1 drug resistance testing before receiving ART,which could be conducive to the choice of clinical drugs.In the second part,in order to evaluate the prevalence of low-frequency resistance mutations in HIV-infected individuals in China,52 treatment-na?ve patients and 21 patients with virologic failure were recruited and performed HIV-1 drug resistance testing by Sanger-based sequencing and deep sequencing.Transmitted drug resistance mutations in PR,RT,and IN were detected in 36.5% and 25.0% of treatment-naive patients by deep sequencing and Sanger-based sequencing,respectively.More low-frequency resistance mutations were detected by deep sequencing than by Sanger-based sequencing in 15.4%(8/52)of the treatment-naive patients.In the virological treatment failure group,drug resistance mutations were detected in 90.5% and 85.7% of patients by deep sequencing and Sanger-based sequencing,respectively.More low-frequency resistance mutations were detected by deep sequencing than by Sanger-based sequencing in 74.1%(15/21)of the patients with virologic failure.The prediction results of drug resistance indicated that HIV-1 drug resistance testing by Sanger-based sequencing may lead to underestimation of high-level resistance to NRTIs and NNRTIs in some patients with virologic failure.If VIII economic conditions permitted,we recommended that the direct use of deep sequencing to detect HIV-1 resistance mutations in patients with virologic failure,which provided support for precise guidance of clinical medication.In the third part,the present study aimed to use the CRISPR/Cas9 system combined with dual single guide RNAs(sg RNAs)to attack HIV-1 DNA in a cell model of latency and to assess off-target effects using whole-genome sequencing.First,we designed 12 sg RNAs targeting HIV-1 DNA in the J-Lat 10.6 cell line,and twelve cell lines stably expressing Cas9 and one sg RNA were constructed using lentiviral vectors.The mutation frequency caused by the CRISPR/Cas9 nuclease cleavage was detected using the T7E1 assay to select effective sg RNAs.Then,according to the results of the T7 EI assay,sg RNAs with relatively high efficiency were selected and nine dual-sg RNA combinations were constructed in the lentiviral vector.The sg RNA target sites in these J-Lat 10.6 cells stably expressing Cas9 dual-sg RNAs were sequenced to analyze the types and the frequencies of the mutations by TA clone.At last,three combinations of dualsg RNAs/Cas9 with high editing efficiency were screened successfully from multiple combinations.Among these combinations,the incidence of insertions and deletions in the sg RNA-targeted regions reached 75.0% and above,and no credible off-target sites were detected using whole genome sequencing.In summary,this study draws the following conclusions:1.The distribution of HIV-1 genotypes in Zhejiang province was dominantly CRF07＿BC and CRF01＿AE,and many other subtypes co-existed.The transmitted drug resistance level in Zhejiang province was high,especially resistance to NRTIs and NNRTIs.Therefore,it is necessary for treatment-naive HIV-infected individuals to perform HIV-1 drug resistance testing before receiving ART.2.Low-frequency resistance mutations were detected by deep sequencing in the patients with HIV-1 who experienced virologic failure and the treatment-na?ve patients with HIV-1.The incidence of low-frequency resistance mutations in patients with virologic failure was high.If economic conditions permitted,we recommended that the direct use of deep sequencing to detect HIV-1 resistance mutations in patients with virologic failure,which provided support for precise guidance of clinical medication.3.We selected three combinations of dual-sg RNAs/Cas9 that targeted HIV genomic DNA with high editing efficiency and no reliable off-target sites were detected using whole genome sequencing.The study provided a new idea and methodological suggestion for CRISPR/Cas9-mediated individualized treatment of HIV-1 in the future. |
