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In this study, researchers used CRISPR gene editing to map genes that play a vital role in erythropoiesis, which may be instrumental in managing aplastic anemia.

The objective of this study, shared in a paper at the 65th ASH Annual Meeting & Exposition, was to understand genes governing red blood cell (RBC) development. While prior studies have focused on external signaling molecules that influence the intracellular processes responsible for erythrocyte production, a substantial gap has remained regarding the specific genes that are essential for successful erythropoiesis. 

Study Used CD49d Expression to Separate Differentiated From Undifferentiated Cells

The study employed a genome-scale CRISPR knock-out screen in the human erythroid progenitor cell line HUDEP-2, utilizing the GeCKOv2 lentiviral library. This library delivers Cas9 along with one of six single-guide RNAs (sgRNAs) targeting nearly every gene in the genome. Through this approach, the researchers aimed to define the range of genes essential for human erythroid development. Cells were collected both before the onset of differentiation and at the differentiated orthochromatic erythroblast stage, based on CD49d expression—a marker known to be downregulated in the final stages of erythroid differentiation.

Gene Knock-Out Confirms Requirement of Specific Genes for Erythroid Development

The findings of the study confirmed the expected depletion of sgRNAs targeting known erythroid essential genes in HUDEP-2 cells compared to the comprehensive sgRNA library, thus effectively validating the efficacy of the chosen methodology. Furthermore, the researchers identified specific genes for which sgRNAs were depleted in differentiated versus undifferentiated erythroid cells, signifying they are probably required for terminal erythroid differentiation. Notably, among the identified genes, NHLRC2, previously implicated in hemolytic anemia, emerged as being intrinsically required for human erythroid development.

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 Over 500 Genes That Influence Erythroid Differentiation Were Identified 

To validate these results, the researchers downregulated NHLRC2 in primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) undergoing differentiation. This intervention resulted in the impairment of both proliferation and differentiation of erythroid cells in vitro. 

To validate the results of the genome-scale screen a secondary library was generated focusing on genes that positively or negatively regulate the final stages of erythroid differentiation while intentionally excluding common essential genes necessary for the survival of the majority of immortalized cell lines screened by the Broad Institute. This secondary screen yielded a comprehensive list of over 500 genes found to influence erythroid differentiation.

Among the novel genes identified in this secondary screen, VAC14 stood out. Validation experiments were conducted, conclusively demonstrating that VAC14 down-regulation, using one of three independent shRNAs, led to significant defects in erythroid cell proliferation. These defects were observed not only in HUDEP2 cells but also in erythroid cells derived from primary human HSPCs. 

To assess the in vivo relevance of the identified genes, the researchers conducted a transplantation experiment. Fetal livers from Vac14-null mice were transplanted into wild-type recipients. Mice receiving Vac14 null hematopoietic stem cells exhibited a profound reduction in the absolute numbers of bone marrow erythroid cells, with evidence of a block in erythroid maturation. 

The Bottom Line

These findings contribute significantly to our understanding of the genetic determinants governing red blood cell development, providing a foundation for further research into potential therapeutic interventions for conditions related to erythroid dysfunction. 

Source

Myers, G. (2023, December 11). Whole-Genome CRISPR-CAS9 screening identifies genes required for human and mouse erythroid development. https://ash.confex.com/ash/2023/webprogram/Paper181929.html