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

Area of Science:

• Genetics
• Molecular Biology
• Protease Function

Background:

• Functional redundancy and gene clustering of homologous genes complicate reverse genetics studies in animal models.
• Generating multigene-deficient animals is challenging using standard breeding techniques.

Purpose of the Study:

• To develop and apply an iterative, multiplexed CRISPR-based approach for simultaneous gene editing.
• To create a comprehensive library of congenic mouse strains deficient in combinations of HAT/DESC proteases.
• To investigate the physiological roles of the HAT/DESC protease gene cluster.

Main Methods:

• Iterative, multiplexed CRISPR-mediated gene editing was employed.
• Four cycles of targeting were performed to generate diverse knockout combinations.
• A library of 18 unique congenic mouse strains, including a complete knockout, was created.

Main Results:

• The generated mouse strains confirmed that HAT/DESC proteases are dispensable for development, postnatal health, and fertility.
• The unique role of HAT-like 4 protease in epidermal barrier formation was validated.
• The study successfully generated a valuable resource for studying HAT/DESC proteases.

Conclusions:

• Iterative, multiplexed CRISPR editing is effective for analyzing multigene clusters.
• The HAT/DESC protease family does not play essential roles in basic physiological processes.
• New mouse models are available for in-depth investigation of HAT/DESC protease functions.