Multiplexed tat-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape

Multiplexed tat-Targeting CRISPR-Cas9 Protects T Cells from Acute HIV-1 Infection with Inhibition of Viral Escape

Blog Article

HIV-1 cure strategy by means of proviral knock-out using CRISPR-Cas9 has been hampered by the emergence of viral resistance against the targeting guide RNA (gRNA).Here, we proposed multiple, concentrated gRNA attacks against HIV-1 regulatory genes to block viral escape.The T cell line were transduced with single and multiple gRNAs targeting HIV-1 tat and rev using lentiviral-based CRISPR-Cas9, followed by replicative HIV-1NL4-3 challenge in vitro.Viral p24 rebound was observed for almost all gRNAs, but multiplexing three tat-targeting gRNAs maintained p24 suppression and cell viability, indicating the inhibition of viral escape.

Multiplexed tat gRNAs Evening gowns inhibited acute viral replication in the 2nd round of infection, abolished cell-associated transmission to unprotected T cells, and maintained protection through 45 days, post-infection (dpi) after a higher dose of HIV-1 infection.Finally, we describe here for the first time the assembly of all-in-one lentiviral vectors containing three and six gRNAs targeting tat and rev.A single-vector tat-targeting construct shows non-inferiority SUPER ONCEA DAY to the tat-targeting multi-vector in low-dose HIV-1 infection.We conclude that Cas9-induced, DNA repair-mediated mutations in tat are sufficiently deleterious and deplete HIV-1 fitness, and multiplexed disruption of tat further limits the possibility of an escape mutant arising, thus elevating the potential of CRISPR-Cas9 to achieve a long-term HIV-1 cure.