Molecular screening of Vel‐blood donors using DNA pools in Nanjing, China

Liu, T. X.; Liu, Y. C.; Ma, Ling; Zhao, Fei; Zhang, R. Y.; Shi, Lili

doi:10.1111/tme.12460

Cited by 1 publication

(1 citation statement)

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“…In several studies in China, researchers identified 2 individuals with Vel− phenotype out of 6,153 donors in Shanghai [ 8 ]. One individual out of 9,122 donors in Jiangsu and 14 individuals out of 3,328 donors in Xinjiang were identified with a heterozygous c.64_80del allele [ 9 , 10 ]. Although the presence of the Vel antigen has important clinical implications, the availability of anti-Vel antibody reagents and red blood cells (RBCs) from Vel− individuals is currently limited.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-Editing K562 Cell Line as a Potential Tool in Transfusion Applications: Knockout of Vel Antigen Gene

Yang,

Li,

et al. 2023

Transfus Med Hemother

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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 knockout (KO) cell lines at present. Methods: To identify the high-efficiency small-guiding RNA (sgRNA) sequence, candidate sgRNAs were transfected into HEK 293T cells and analyzed using Sanger sequencing. Following this, the high-efficiency sgRNA was transfected into K562 cells using lentivirus transduction to generate KO Vel blood group gene cells. The expression of the 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 Vel erythroid antigen KO single-cell clones in K562 cells. The edited cells could then be induced to undergo erythroid differentiation with the use of hemin.

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Section: Introductionmentioning

confidence: 99%