Figure 3
From: Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9
HIV-1 strain R7 replication in CRISPR human CD4+ T cell lines.
Wild type human CD4+ T cell line SupT1 and derivatives expressing Cas9 and multiple gRNAs targeting HIV-1 were infected with strain R7. Compared to NL4-3, R7 is known to have slower growth kinetics. (Left) Culture supernatants were assayed for the release of p24 as a measure of viral replication. (Right) Cell viability was measured by trypan blue exclusion. As viral replication increases, cellular viability decreases. Error bars indicate the standard deviation between three separate infections. HIV-1 R7 infection of the gRNA-TATA1 cell line was included as a negative control. The R7 strain encodes one mismatch from gRNA-TATA1 7 bp distal to the PAM. While Cas9 has been shown to tolerate limited mismatches for some gRNAs, empirically this single mismatch appears sufficient to prevent CRISPR/Cas9 gRNA-TATA1 editing and viral replication was equal to wild type cells.
