
 Fig . 2. Generation of p38-α- and p38-Υ knockout Vero cells. sgRNAs targeting (at least four sgRNAs per target gene) p38-α (MAPK14) or p38-Υ (MAPK12) were cloned in pL-CRISPR.EFS.GFP and sequenced. Vero cells were transfected with the constructs expressing sgRNAs. At 48 h post transfection, cells were trypsinized and subjected to sort the GFP expressing cells by FACS. The GFP expressing cells were cultured in 96 well plates by limiting dilution assay. Wells with a single clone were identified and further cultured (11 clones of each gene). To validate editing, three to four clones were subjected to PCR amplification and nucleotide sequencing of the target gene, besides evaluating the expression levels of the target protein by Western blot analysis. ( A ) Amplification of p38-α (MAPK14) gene from three (α-3, α-6, and α-10) selected clones of p38-α KO cells. ( B ) Nucleotide sequencing depicting deletion of 113 bp in clone α-3. ( C ) Amplification of p38-Υ (MAPK12) gene from four (Υ-1, Υ-2 Υ-3 and Υ-4) selected clones of p38-Υ KO cells. ( D ) Nucleotide sequences depicting deletion of 4 bp in clone Υ-4. ( E ). Expression of p38-α in WT, p38-Υ KO, and p38-α KO cells by Western blot analysis. ( F ) Expression of p38-Υ in WT, p38-α KO, and p38-Υ KO cells by Western blot analysis.