| Summary: | 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 <i>tat</i> and <i>rev</i> using lentiviral-based CRISPR-Cas9, followed by replicative HIV-1<sub>NL4-3</sub> challenge in vitro. Viral p24 rebound was observed for almost all gRNAs, but multiplexing three <i>tat</i>-targeting gRNAs maintained p24 suppression and cell viability, indicating the inhibition of viral escape. Multiplexed <i>tat</i> gRNAs 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 <i>tat</i> and <i>rev</i>. A single-vector <i>tat</i>-targeting construct shows non-inferiority to the <i>tat</i>-targeting multi-vector in low-dose HIV-1 infection. We conclude that Cas9-induced, DNA repair-mediated mutations in <i>tat</i> are sufficiently deleterious and deplete HIV-1 fitness, and multiplexed disruption of <i>tat</i> further limits the possibility of an escape mutant arising, thus elevating the potential of CRISPR-Cas9 to achieve a long-term HIV-1 cure.
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