The overall objective of this application is the development of a mutant cell library that will lead to the validation and characterization of the structure-function relationship (SFR) of heparan sulfate (HS) in a cellular context. HS abundantly expresses at the cell surface and in the extracellular matrix where it interacts with a large number of protein ligands, including growth factors, growth factor receptors, morphogens, cytokines and matrix proteins to modulate a variety of physiological and pathological processes such as development, leukocyte trafficking, tumorigenesis, angiogenesis and lipid metabolism. Understanding in depth the biological functions and related structure of HS represents one of the most active and challenging studies in the glycoscience field. Biochemical studies using oligosaccharides have established that, in general, HS requires distinct structures to interact with different proteins. However, the SFR of HS in a cellular context is far more complex. This in part results from the fact that natural HS is much larger than the oligosaccharides used in biochemical analyses, may interact simultaneously with both a ligand and the ligand`s receptor via distinct binding sites, and may also harbor multiple copies of the binding sites. The content and geometry of ligand/receptor-binding sites within the HS chain are also believed to critically modulate HS-ligand/receptor interactions, representing a higher level of regulatory mechanism. Development of a cell library that contains all the potential HS structure alterations in nature to extend and validate the biochemical findings in a cellular context is essential for a better understanding of the biological functions of HS in vitro and in vivo. Expanding our studies on conditional gene deletion-derived HS mutant mouse lung endothelial cells (MLEC), in this application we propose using the novel, highly efficient CRISPR-Cas9 gene editing technology to develop a HS mutant MLEC library that includes all the major HS structure alterations in nature to enable systematic interrogation of HS SFR in a cellular context. For this purpose, we will pursue two Specific Aims: 1. Generation of a HS mutant cell library by targeting individual or combination of HS biosynthetic genes that are expressed in MLEC lines; 2. Validation of the generated mutant cell library for HS SFR studies by examining cells at cell surface binding and cell responses upon stimulation with ligands that require unique HS modification(s) for their binding. Novel and established genetic, cellular and biochemical approaches in conjunction with in vitro cell function studies will be used to develop the proposed HS mutant cell library. These efforts are anticipated to result in a novel, comprehensive, easy to use HS mutant library that enables systematic interrogation of HS SFR in a cellular context and a tool set of CRISPR-Cas9-based highly efficient HS biosynthetic gene targeting reagents. The availability of these reagents is expected to greatly accelerate current HS studies.