Generation of knockout mice for functional validation. a–e Genomic structure and knockout strategy for mouse Spint3 (a), Spint4/5 (b), Ces5a (c), Pp2d1 (d), and Saxo1 (e). Single or dual sgRNAs were designed to target the indicated exons in each gene. Each founder animal’s deletion mutation is indicated with red hash lines. For all but Spint4/5, F1 and R1 are wild-type primers and F2 and R2 are mutant primers. For Spint4/5, F1 of Spint4 was combined with R2 of Spint5 to detect the mutant allele. f–j Representative Sanger sequence result of mutant mice depicted in a–e, respectively. k–o Representative genotype result of mutant mice using the indicated primers in a–e, respectively.