Targeting, Mutagenesis, and Functional Testing Strategies for Large Collagens

Collagens comprise a homogeneous family of cell-surface or extracellular proteins. Many of them are fundamental for health, and consequently, their deficiency or dysregulation occurs in a wide range of diseases, from tissue fragility to neoplastic and fibrosing diseases, to name a few. With the advent of new omics approaches, an increasing number of variants of specific collagens have been discovered in various conditions. Some of these variants lead to a loss of expression, highlighting the need to assess the functions and loss of specific collagens in such conditions, while other variants may involve changes in amino acids of unknown consequences. Thus, with the advent of newer omics technologies, there is an increasing need to engineer specific collagens and their encoding genes. However, for larger collagens, this can be challenging because of their size and repetitive structure. Using collagen VII as an example of a large collagen, we will describe strategies for protein engineering, functional analysis, and collagen gene targeting in cells that naturally express collagens.