Journal
SCIENTIFIC REPORTS
Volume 8, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41598-018-26190-1
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Funding
- Japan Society for the Promotion of Science (JSPS)
- program of the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) from the Ministry of Education, Culture, Sport, Science and Technology in Japan
- Japan Agency for Medical Research and Development (AMED)
- NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH [9849]
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The CRISPR/Cas9 system provides a novel and promising tool for editing the HIV-1 proviral genome. We designed RNA-guided CRISPR/Cas9 targeting the HIV-1 regulatory genes tat and rev with guide RNAs (gRNA) selected from each gene based on CRISPR specificity and sequence conservation across six major HIV-1 subtypes. Each gRNA was cloned into lentiCRISPRv2 before co-transfection to create a lentiviral vector and transduction into target cells. CRISPR/Cas9 transduction into 293T and HeLa cells stably expressing Tat and Rev proteins successfully abolished the expression of each protein relative to that in non-transduced and gRNA-absent vector-transduced cells. Tat functional assays showed significantly reduced HIV-1 promoter-driven luciferase expression after tat-CRISPR transduction, while Rev functional assays revealed abolished gpl20 expression after rev-CRISPR transduction. The target gene was mutated at the Cas9 cleavage site with high frequency and various indel mutations. Conversely, no mutations were detected at off-target sites and Cas9 expression had no effect on cell viability. CRISPR/Cas9 was further tested in persistently and latently HIV-l-infected T-cell lines, in which p24 levels were significantly suppressed even after cytokine reactivation, and multiplexing all six gRNAs further increased efficiency. Thus, the CRISPR/Cas9 system targeting HIV-1 regulatory genes may serve as a favorable means to achieve functional cures.
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