A 20-bp guide sequences targeting the fourth exon of canine HIF2α (Epas1)) and the sixth exon of canine Dock4 were designed online using Zhang’s laboratory web resource (www.genome-engineering.org ). gRNA-encoding oligonucleotides (Sigma-Aldrich) were cloned into the vector SpCas9(BB)-2A-GFP (PX458, Addgene plasmid ID 48138) using standard procedures as described29 (link). The generation of the HIF2α-KO and Dock4-KO cells via CRISPR-Cas9- mediated non-homologous end-joining (NHEJ) DNA repair and the screening were performed according to described guidelines29 (link).
In brief, the MDCK cells were transiently transfected with the genome editing CRISPR-Cas9 construct and 48 h post-transfection cells were subjected to single-cell-sorting. The single-cell clones were expanded and screened for frame-shift mutations; shortly, a region spanning the target site was amplified by PCR from genomic DNA isolated from clonal cell lines. PCR products were subsequently cloned into pUC19 (Invitrogen). 15–20 sequences were selected based on FASTA similarity search-tool (EMBL-EBI) (TableS2 ).
In brief, the MDCK cells were transiently transfected with the genome editing CRISPR-Cas9 construct and 48 h post-transfection cells were subjected to single-cell-sorting. The single-cell clones were expanded and screened for frame-shift mutations; shortly, a region spanning the target site was amplified by PCR from genomic DNA isolated from clonal cell lines. PCR products were subsequently cloned into pUC19 (Invitrogen). 15–20 sequences were selected based on FASTA similarity search-tool (EMBL-EBI) (Table