Figure 1
From: Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9
CRISPR expression does not impair cell growth or lead to significant off-target editing.
(Left) Wild type SupT1 cells and CRISPR cell lines were plated at the same density. Viable cells were counted by trypan blue exclusion. The growth kinetics of the CRISPR expressing cells are similar to wild type cells. (Right) Surveyor assay of off-target editing at the two highest predicted sites in human genes. Positive and negative assay controls were included for each set of reactions. M, marker. The matrix indicates the inclusion of homoduplex DNA (hom), heteroduplex DNA (het), or Surveyor nuclease (nuc) for all the gels below. Lanes with Surveyor assay controls are indicated (control) and off-target sites are indicated by the name of the site in white text. Lanes 1 and 2, Surveyor negative control homoduplex shows background cleavage activity as a faint smear. Lanes 3 and 4, Surveyor positive control heteroduplex shows cleavage products as two faster mobility bands. Lanes 5 and 6, off-target 1 PCR products from wild type cells. Lanes 7 and 8, off-target 1 PCR products from wild type cells annealed to PCR products from CRISPR cells. Lanes 9 and 10, off-target 2 PCR products from wild type cells. Lanes 11 and 12, off-target 2 PCR products from wild type cells annealed to PCR products from CRISPR cells. The absence of faster mobility products in lanes 8 compared to lanes 6 and lanes 12 compared to lanes 10 indicate that off-target editing is minimal in these cells. CRISPR gRNA-TATA1 off-targets are TNK2 and ABHD15. gRNA-TATA2 off-targets are YTHDC1 and CCDC146. gRNA-TAR1 off-targets are CELSR1 and SOX2-OT. gRNA-TAR2 off-targets are LOC146880 and SOX2-OT. gRNA-RRE/env off-targets are WNT8B and EPHA1-AS1.
