Supplementary Material for: CRISPR/Cas9-editing K562 cell line as a potential tool in transfusion applications: Knockout of Vel Antigen Gene

Introduction: The Vel- phenotype is a rare blood group, and it is challenging for identifying this phenotype due to limited available reagents. Moreover, there are relatively few studies on genomic editing of erythroid antigens and generation of KO cell lines at present. Methods: To identify the high efficiency sgRNA sequence, candidate sgRNAs were transfected into HEK 293T cells respectively and analyzed using Sanger sequencing. Following this, the high efficiency sgRNA was transfected into K562 cells using lentivirus transduction to generate knockout Vel blood group gene cells. The expression of Vel protein was detected using western blot on single cell clones. Additionally, flow cytometry was used to detect the erythroid markers CD235a and CD71. Hemoglobin quantification and Giemsa staining were also performed to evaluate the erythroid differentiation of KO clones induced by hemin. Results: The high efficiency sgRNA was successfully obtained and used for CRISPR-Cas9 editing in K562 cells. After limiting dilution and screening, two KO clones had either deleted 2 or 4 bases and showed no expression of the Vel protein. In the hemin-induced KO clone, there was a significant difference in erythroid marker and hemoglobin quantification compared to untreated cells. The morphological changes were also observed for the hemin-induced KO clone. Conclusion: In this study, a highly efficient sgRNA was screened out and used to generate single-cell clones knockout Vel erythroid antigen in K562 cells. The edited cells could then be induced to undergo erythroid differentiation with the use of hemin.