Figure 6
Knockout of chIFIT5 gene from chicken fibroblasts using CRISPR/Cas9 genome editing technology and evaluation of the influence of chIFIT5 on the replication of NDV and VSV. (A) DF-1 cells were co-transfected with hybridized crRNA and tracrRNA and plasmid expressing both Cas9 and puromycin resistance gene. 48 hours post-transfection; cells were passaged and selected with puromycin (10 ug/ml) until complete death of non-transfected cells. Puromycin-resistant cells were transfected with GFP expressing plasmid and were FACS sorted into individual cells and expanded until desire number of cells was achieved. At least 5 clones were confirmed for editing before clonal expansion and phenotypic analysis. (B) T7EI assays confirmation of the InDels in clonal cells that were supplemented with all essential components of gene editing including crRNA, tracrNA and Cas9 (lane number 3) compared to Cas9 control (lane number 2) and a no-input control (lane number 4). (C) Confirmation of gene editing using DNA sequencing of amplicons spanning the putative InDels site. (D,E) Wild type and chIFIT5KO-confirmed DF-1 cells were infected with NDV (D) or VSV (E). Replication of RNA viruses was monitored for 24 hours of post-infection and shown in fluorescent units. (F) Wild type and chIFIT5KO-confirmed DF-1 cells were infected with NDV or were left uninfected for 24 hours before processing them for FACS. Cumulative MFI of GFP positivity in wild type and chIFIT5KO DF-1 cells based on at least 3 independent experiments. (G) DF-1 cells were either mock transfected (wt DF-1 cells) or were transfected with 2 ug of chIFIT5 expression plasmid (chIFIT5OE DF-1 cells). These cells and CRISPR/Cas9 knockout DF1 (chIFIT5KO) were either left un-infected or were infected with VSV for 48 hours before staining with crystal violet. The plagues developments were imaged using hand-held camera.
