To test for gene editing, a 20-nt gRNA was designed to target a disrupted enhanced green fluorescent protein (EGFP) gene present in a reporter vector. After the gRNA introduced a double-stranded break, cells attempted to repair the cut site via HDR using a DNA template within the reporter vector. In the event of successful gene editing, the EGFP sequence was restored to a functional state and green fluorescence was detectable by flow cytometry. To achieve gene repression, a 14-nt gRNA was designed to target LIGIV. The gRNA included a com protein recruitment domain, which recruited a Com-KRAB fusion protein to facilitate gene repression via chromatin modification of LIGIV. Quantitative polymerase chain reaction was used to quantify repression.
This study expanded upon earlier advancements, offering a novel and versatile approach to genetic modification and transcriptional regulation using CRISPR/Cas9. The overall results show that both gene editing and repression were occurring, thereby providing support for a novel CRISPR/Cas system capable of simultaneous gene modification and regulation. Such a system may enhance the genome engineering capabilities of researchers, benefit disease research, and improve the precision with which gene editing is performed.
CD47 is a cell surface receptor expressed on many cells in the body. It has many immune functions such as marking host cells as “self” and the activation of apoptosis through phagocytosis. Mac-1 is a major integrin on myeloid cells and has been implicated in several different macrophage immune functions. Previous studies from Dr. Ugarova’s lab demonstrated CD47 may form a complex with Mac-1 through the cis-interaction and could regulate Mac-1-dependent macrophage functions. To localize the binding site for Mac-1 in CD47, the extracellular domain of CD47 IgV was isolated as GST-fusion protein from E. coli cells. The recombinant fusion protein is being used in current studies with cell adhesion assays and immunoprecipitation to determine the complementary binding site in Mac-1.