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

Abstract

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.

• Authors: LuLu K Callies, Daniel Tadeo, Jan Simper, Thomas H Bugge, Roman Szabo
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 294 Issue 44 Pg. 15987-15996 (11 01 2019) ISSN: 1083-351X [Electronic] United States
• PMID: 31501243 (Publication Type: Journal Article, Research Support, N.I.H., Intramural, Research Support, Non-U.S. Gov't)
• Copyright: © 2019 Callies et al.

Chemical References

• Serine Endopeptidases
• hepsin

Topics

• Animals
• CRISPR-Cas Systems
• Embryonic Development (genetics)
• Epidermis (growth & development, metabolism)
• Female
• Fertility (genetics)
• Gene Editing (methods)
• HEK293 Cells
• Humans
• Male
• Mice
• Multigene Family
• Serine Endopeptidases (genetics, metabolism)