The CRISPR/Cas9 system provides a new potential tool for editing the HIV-1 viral genome. Recently, researchers from the Department of Infectious Diseases of the Medical College of Kobe University of Japan and the Department of International Health Sciences of the Graduate School of Health Sciences designed an RNA-directed CRISPR/Cas9 to target HIV-1 regulatory genes tat and rev, including guide RNAs (gRNA). Both are based on the specific design of CRISPR, and their targeted sequences are conserved among the six major HIV-1 subtypes.
Each gRNA was cloned into the CRISPRv2 lentivirus prior to co-transfection, creating a lentiviral vector and transducing it into the cell. The expression of both genes in the cells was successfully inhibited by CRISPR/Cas9 transfection of 293T and HeLa cells stably expressing Tat and Rev compared to cells transfected and transfected with empty vector.
Tat function assay showed that transfection of tat-C CRISPR significantly inhibited the expression of luciferase driven by HIV-1 promoter, while Rev function assay showed that gp120 expression was completely inhibited after transfection of rev-CRISPR. The target gene of the Cas9 cleavage site exhibits various degrees of high frequency mutations. It is worth noting that the researchers did not detect any mutations in the target site, and the expression of Cas9 had no effect on cell viability.
The researchers further tested their CRISPR/Cas9 system in HIV-1 infected T cell lines and found that even with cytokine re-stimulation, p24 expression was significantly inhibited, while the use of six gRNAs could further enhance editing. effectiveness. Therefore, targeting HIV-1 regulatory genes using the CRISPR/Cas9 system may be an effective way to achieve functional healing.
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