Identification of Novel Regulators of Erythropoiesis Using Whole-Genome CRISPR-Cas9 Screening

Red blood cell (RBC) development is regulated by a few external signaling molecules which act to influence the ongoing intracellular processes responsible for producing erythrocytes. While much is known about the signaling molecules important for blood production, comparatively little is known about the genes required for successfully generating erythrocytes. Several genes have already been identified as essential for RBC development through loss-of-function studies in the mouse or in human erythroid cells in vitro. However, these studies have been limited in scope to genes that are already suspected or known to influence RBC production. Since there are ~10,000 genes expressed in erythroid cells, it is unclear how many of these genes are functionally required for erythroid development.

To define the repertoire of genes required for human erythroid development, we have performed a genome-scale CRISPR knock-out screen in the immortalized erythroid progenitor cell line HUDEP-2, using the GeCKOv2 lentiviral library, which delivers Cas9 and one of 6 sgRNAs targeting virtually every gene in the genome. Following viral transduction, we collected HUDEP-2 cells prior to the onset of differentiation as well as differentiated erythroid cells (right prior to enucleation) using flow sorting, on the basis of CD49d expression (CD49d is downregulated in the final stages of erythroid differentiation).

As expected, sgRNAs targeting known erythroid essential genes (such as GATA1 and EPOR) were depleted in HUDEP-2 cells compared to the sgRNA library. Notably, we also identified genes for which sgRNAs were depleted at the end of differentiation (but not before differentiation), representing genes that are likely required for terminal erythroid differentiation (such as ZFPM1, ALAS2, etc..). This supports the utility of the screen to identify novel regulators of erythropoiesis. Based on the results of this primary screen, we then generated a secondary library dedicated to validating genes that positively or negatively regulate the final stages of erythroid differentiation, while excluding common essential genes required for the survival of more than 90% of immortalized cell lines screened by the BROAD institute (DepMap.org).

The secondary screen yielded a list of over 500 genes that are required for erythroid differentiation (FDR<0.01). Of these genes, only 45 were predicted to influence the erythroid count by genome-wide association studies (GWAS) (https://www.ebi.ac.uk/gwas), suggesting that CRISPR screens provide an opportunity to identify novel genes required for erythroid development, beyond those nominated by GWAS. Several of the top ranked genes in the screen have documented roles in erythropoiesis; however, these only represent approximately 25% of the top 100 genes. Genes such as PPP1R21 and GMDS ranked highly in the secondary screen and have GWAS associations with erythroid traits; therefore, these genes have a high probability of influencing human erythroid development. Other highly ranked genes, such as FBXO28 and CNOT4, have no prior documented role in erythropoiesis and therefore represent novel candidate genes that regulate erythroid differentiation. We are currently validating and characterizing the impact of these and other novel candidate genes on terminal erythroid maturation in HUDEP-2 and CD34+ cells differentiated into erythroid cells in vitro.

Engel:Poseida Therapeutics: Consultancy; Imago Biosciences: Consultancy, Current equity holder in publicly-traded company; Fulcrum Therapeutics: Consultancy.