The kallikrein-related peptidases (KLKs) appear to be involved in many physiological and pathological processes, however their roles in vivo are still not fully understood, partially due to the unavailability of suitable mouse models. KLK5 and KLK7 have been identified to be involved in the tightly regulated proteolytic pathways that are crucial for epidermal homeostasis. Knock-out mouse models for KLK5 and KLK7 did not show an obvious phenotype and generation of a mouse model lacking both proteases by conventional strategies is impossible as both genes are located within close proximity on the same locus. Thus, double-deficient KLK5/KLK7 mice were generated using the microinjection of TALEN mRNA targeting Klk7 into Klk5-deficient oocytes. These KLK5/7 double deficient mice show severe phenotype in altered barrier integrity and peculiarly strong thickening of the skin, caused by defective shedding of keratinocytes from the skin surface. These results provide in vivo evidence that KLK5 and KLK7 together are indispensable for the process of desquamation.