Iterative, multiplexed CRISPR-mediated gene editing for functional analysis of complex protease gene clusters

Abstract

Edited by George N. DeMartino Elucidation of gene function by reverse genetics in animal models frequently is complicated by the functional redundancy of homologous genes. This obstacle often is compounded by the tight clustering of homologous genes, which precludes the generation of multigene-deficient animals through standard interbreeding of single-deficient animals. Here, we describe an iterative, multiplexed CRISPR-based approach for simultaneous gene editing in the complex seven-member human airway trypsin-like protease/differentially expressed in a squamous cell carcinoma (HAT/DESC) cluster of membrane-anchored serine proteases. Through four cycles of targeting, we generated a library of 18 unique congenic mouse strains lacking combinations of HAT/DESC proteases, including a mouse strain deficient in all seven proteases. Using this library, we demonstrate that HAT/DESC proteases are dispensable for term development, postnatal health, and fertility and that the recently described function of the HAT-like 4 protease in epidermal barrier formation is unique among all HAT/DESC proteases. The study demonstrates the potential of iterative, multiplexed CRISPR-mediated gene editing for functional analysis of multigene clusters, and it provides a large array of new congenic mouse strains for the study of HAT/DESC proteases in physiological and in pathophysiological processes.

Proteolytic Activation of Plant Membrane-Bound Transcription Factors

“…A genetics approach is also hampered by potential redundancy between proteases that can cleave one MB-TF ( Iwata et al, 2017 ). Therefore, a CRISPR-Cas library screen targeting multiple proteases simultaneously, in random combinations, may be a useful alternative ( Callies et al, 2019 ). A more direct method for the detection of the responsible protease is chemical genomics using a chemical protease inhibitor as a bait.…”

Intracellular autoactivation of TMPRSS11A, an airway epithelial transmembrane serine protease

“…Most of the Tmprss11 genes are expressed in multiple epithelial tissues, including the trachea, esophagus, stomach, bladder, and skin (51,52). Tmprss11a-deficient mice, however, did not exhibit noticeable defects in embryonic development and postnatal survival (51,53). In humans, TMPRSS11A is expressed in the trachea (27,28).…”

Membrane-anchored serine proteases as regulators of epithelial function

“…The rate of fluid loss is relatively small compared with that observed in mice lacking either matriptase or prostasin, and it does not affect postnatal development or long-term survival of Tmprss13-or Tmprss11f-deficient mice, suggesting that at least in the absence of additional challenge, the individual contribution of the two proteases to the establishment of the epidermal barrier is relatively minor. Loss of TMPRSS11F did not alter the histological appearance of the epidermis, including stratum corneum, or the expression of any of the major components of the cornified envelope, whereas newborn Tmprss13-deficient mice exhibit reduced stratum corneum thickness, but no obvious defects in TJ function or profilaggrin processing analogous to the ones observed in matriptase-and prostasin-deficient epidermis [44,48]. It is, therefore, unclear whether the two proteases contribute to the formation of epidermal barrier function either in parallel or as one of several functionally relevant targets acting downstream of the matriptase/prostasin system.…”

“…Both proteases are expressed in a wide array of mouse and human tissues, with high levels detected in keratinized epithelia of skin, oral cavity and upper digestive system [43][44][45][46]. Both Tmprss13-and Tmprss11f-deficient newborn mice present with significantly increased transepidermal fluid loss, indicative of compromised epidermal barrier function [44,47,48]. The rate of fluid loss is relatively small compared with that observed in mice lacking either matriptase or prostasin, and it does not affect postnatal development or long-term survival of Tmprss13-or Tmprss11f-deficient mice, suggesting that at least in the absence of additional challenge, the individual contribution of the two proteases to the establishment of the epidermal barrier is relatively minor.…”