Insights into the mechanisms underlying the inactivation of HIV-1 proviruses by CRISPR/Cas

Abstract DNA editing using CRISPR/Cas has emerged as a potential treatment for diseases caused by pathogenic human DNA viruses. One potential target is HIV-1, which replicates via a chromosomally integrated DNA provirus. While CRISPR/Cas can protect T cells from de novo HIV-1 infection, HIV-1 frequently becomes resistant due to mutations in the chosen single guide RNA (sgRNA) target site. To address this problem, we asked whether an sgRNA targeted to a conserved, functionally critical HIV-1 sequence might prevent the selection of escape mutants. We report that two sgRNAs specific for the HIV-1 transactivation response (TAR) element produce opposite results: the TAR2 sgRNA rapidly selects for mutants that retain TAR function, but are no longer inhibited by Cas9, while the TAR1 sgRNA fails to select any replication competent TAR mutants, most probably because it is targeted to a region of TAR that is disrupted by even minor mutations.