Identifying Novel Regulators of Erythroid Maturation and Proliferation Using a Genome-Scale CRISPR Activation Screen

Red blood cells (RBCs) provide the essential function of oxygen delivery to the body's tissues and organs. Though RBC transfusion is a lifesaving therapeutic intervention, RBCs are a resource often in shortage, particularly in lower-income countries. This is especially true considering that 47% of blood donations are collected in high-income countries, which house only 19% of the global population. Furthermore, the RBC shelf life is ~6 weeks; therefore, even regions with adequate stocks could readily enter periods of shortage. To alleviate these shortages, the development of a methodology for the efficient, scalable, and safe production of RBCs ex vivo is needed.

To identify novel genes that enhance erythroid differentiation/ proliferation, we performed a functional pooled genome-wide CRISPR activation (CRISPRa) screen using the human erythroid cell line HUDEP-2. In this screen, we obtained the CRISPRa v2 library (Addgene: #1000000078), expanded it in competent bacterial cells, isolated pooled plasmids, and confirmed adequate expansion of the library without significant drifts in small guide RNAs (sgRNAs). Subsequently, we prepared pooled lentiviral stocks from the expanded plasmid library. This viral library delivers a VP64 activation domain fused to a catalytically dead Cas9 (dCas9-VP64), a blasticidin resistance cassette, and one of 3 unique sgRNAs targeting virtually every coding gene in the human genome (the library consists of 70,290 sgRNAs). In parallel, we generated a clonal HUDEP-2 cell line that stably expresses the transcriptional activator complex MPH (MS2-P65-HSF1), hereafter referred to as HUDEP-2-MPH cell line. We confirmed adequate growth and differentiation of the latter cell line, as well as validated MPH expression/function.

The HUDEP-2-MPH clonal cell line was subsequently transduced at a low multiplicity of infection (~0.3) with the lentiviral CRISPRa library. Transduced cells were selected and subsequently underwent synchronized erythroid differentiation. Cells were harvested at 2 time points: 1) D0 of differentiation (corresponding to proerythroblasts) and 2) D10 of differentiation, sorting for orthochromatic erythroblasts using cell surface markers. We harvested genomic DNA from both populations, sequenced the sgRNAs using NextGen sequencing, and compared the abundance of the sgRNAs between both cell populations. This experiment was done in biological triplicates, while maintaining >200x sgRNA coverage at all times.

As expected, the screen identified known regulators of erythroid differentiation and/or proliferation, including EPOR and KIT, confirming the validity of the screen. Notably, this screen also identified several novel candidate genes that appear to either promote or reduce the efficiency of terminal erythroid maturation and/or proliferation. The top ranked gene was IGF2BP3, which encodes the Insulin-like growth factor 2 mRNA-binding protein 3.

In preliminary validation studies, we used 3 independent sgRNAs to activate IGF2BP3 in HUDEP-2-MPH cells. Compared to cells transduced with control sgRNAs, increased expression of IGF2BP3 mRNA (by ~20 fold) resulted in a 3-fold increase in cell expansion during differentiation. Additionally, HUDEP-2-MPH cells transduced with IGF2BP3 activating sgRNAs appeared to exhibit enhanced differentiation capacity, with ~67% of cells displaying Band3 expression and CD49d downregulation at day 12 of differentiation (compared to ~30% of cells transduced with control sgRNAs). These findings were further confirmed by morphologic evaluation of cytospins prepared from cells at day 12 of differentiation.

We are currently further validating the impact of increased IGF2BP3 expression on erythroid differentiation in CD34+ cells differentiated into erythroid cells in vitro. Additionally, we are evaluating the role of other novel genes (identified in our screen) on erythroid maturation and proliferation. Targeting key genes found in this screen is anticipated to improve the efficiency of erythroid maturation. These findings have important implications for the ability to generate sufficient numbers of mature RBCs ex vivo, which could be used to balance the worldwide supply and demand of RBCs for transfusions.

No relevant conflicts of interest to declare.