Fig. 1

Construction of CRISPR/Cas9-mediated canine TP53 gene knockout (KO) system. a Procedure to design and select guide RNAs (gRNAs) specifically targeting canine TP53. Step 1: Candidate gRNAs for all exons of the gene were searched using the CRISPR Target Finder Program. Step 2: Candidates for 1–3 exons were selected to avoid partial functional protein production. Steps 3 and 4: gRNAs with less binding potential to the canine genome and transcripts were sorted according to the BLAST alignment algorithm. The final three gRNAs of TP53 were inserted into the CRISPR/Cas9 expression vector. b Sequences of the three gRNAs targeting canine TP53 exon 3. c Transient transfection and Cas9 gene expression was detected based on enhanced green fluorescence proteins (EGFP) reporter transgene, which was cleaved from the Cas9 protein via 2A peptide-mediated self-cleavage