Generating Human Cell Lines Deficient in SEC23A and SEC23B to Study the Pathophysiology ofCDAII and ER to Golgi Protein Transport

Science Center, Bent Corridor

Congenital Dyserythropoietic Anemia type II (CDAII) is a bone-marrow intrinsic autosomal recessive disease characterized by abnormal erythroid maturation that results from mutations in SEC23B. SEC23 is a component of COPII vesicles, which transport secretory proteins from ER to Golgi. Mammals have two paralogs for SEC23, SEC23A and SEC23B. The goal of this study is to better understand the pathophysiology of CDAII by establishing/characterizing an in-vitro model using genome editing with CRISPR/Cas9. To determine the impact of SEC23B and SEC23A deficiency on erythropoiesis, human erythoid cell lines (K562 and HUDEP-2) were subjected to genome editing with CRISPR/Cas9 using various gRNAs targeting SEC23A and SEC23B. Single-cell clones were generated that are being evaluated for in/dels and will be further analyzed for defects in erythroid development morphologically (light microscopy) and using flow cytometry. We have already generated 293T-REx cells that express the SEC23A (or SEC23B) cDNA under the control of a tetracycline-inducible promoter. We deleted the endogenous SEC23A and SEC23B genes in these cells using gRNAs that target exon-intron junctions. 11 single-cell clones appear to have on-target modifications for both genes. If upon removal of tetracycline, cells deficient in SEC23 survive, we will compare the secretome of these cells to that of WT cells. We predict that cells lacking COPII vesicles are not viable, in which case, we will titrate the expression of SEC23A or SEC23B to the lowest level, and compare the secretome of these cells to that of WT cells to determine whether any proteins can bypass the COPII secretory pathway.