UC Berkeley

The vital roles of red blood cells (RBCs) underscore the importance of functional and homeostatic cellular processes in erythroid precursors. Iron acquisition and trafficking by erythroid cells is a key physiological process for their survival, proliferation and differentiation. In contrast, exposure to hematotoxicants can deplete erythroid precursors or disrupt erythroid differentiation. Complete elucidation of mechanisms relevant to a biological process or response to an injury in erythroid cells can improve our understanding of the pathophysiology and etiology of RBC disorders. Interrogating the genome in the context of a cellular process is a key strategy to understand underlying molecular mechanisms. Comprehensive identification of pathways governing physiological as well as injurious cellular processes has become more feasible due to the emergence of genome-wide functional genomic approaches. Recently, the CRISPR-Cas9 system has revolutionized the field of functional genomics as it enabled simple, efficient and cost-effective platforms for large-scale genetic screening. In this series of studies, CRISPR-based genome-wide loss-of function screening was employed to identify genes and pathways contributing to erythroid key processes including iron uptake, heme trafficking and erythroid differentiation, as well as response to hematotoxicants.

Chapter 1 reviews the various CRISPR-Cas9 screening platforms and their applications in functional genomics. Different screening strategies using large-scale loss-of-function and gain-of-function approaches are compared in terms of their effectiveness, feasibility, and suitability to study particular cellular processes. Steps of pooled screening, data analysis methods and hit validation strategies are described and critical technical considerations are addressed. In addition, the power of CRISPR-based genetic screening in different research fields including basic cell biology, cancer, drug discovery, pharmacogenomics and toxicogenomics is demonstrated. Finally, limitations of the current CRISPR screening platforms are revised and strategies for improvement are proposed.

In chapter 2, a genome-wide CRISPR-based screen designed to study iron uptake in erythroid cells is described. The physiological iron form, transferrin-bound iron (TBI), is taken up by erythroid cells through the transferrin receptor (TfR) by endocytosis. Non-transferrin bound iron (NTBI), which is present in the circulation during iron overload, can also be acquired by erythroid cells. Uncontrolled cellular NTBI influx can result in iron toxicity. To identify molecular determinants of TBI and NTBI uptake, a genome-wide loss-of-function screen was performed in human K562 erythroleukemic cells which can utilize both TBI and NTBI to grow and proliferate. The screen revealed multiple genes whose disruption resulted in defective growth when either TBI or NTBI is the exclusive source of iron. Unsurprisingly, TBI uptake candidates included the transferrin receptor (TfR1) in addition to several components of the endocytic pathway. Follow up studies on one candidate, CCDC115, confirmed its role in cellular TBI uptake. CCDC115 is a V-ATPase assembly factor and its role in endosomal acidification likely underlies its function in transferrin iron influx.

In chapter 3, a genome-wide knockout screen investigating cellular mechanisms of heme trafficking and heme-induced erythroid differentiation is documented. Under physiological conditions, heme is synthesized in the mitochondria of erythroid cells where it plays structural as well as functional roles in erythroid differentiation. Additionally, erythroid cells are capable of taking up extracellular heme which might be utilized in pathological forms of erythropoiesis or could induce toxicity in the absence of other erythropietic signals. Heme treatment of K562 erythroleukemic cells induces erythroid differentiation resulting in a proliferation block. The screen was designed to identify genes whose inactivation alleviates the heme-induced proliferation block. The identified candidates include components of the clathrin-mediated endocytosis and vesicle acidification pathways in addition to epigenetic and RNA processing regulators. Intriguingly, the V-ATPase assembly factor CCDC115 was among the top candidates. Analysis of CCDC115 deficient cells unveiled an unprecedented role of CCDC115 in cellular heme uptake. Gene products whose loss results in heme-induced toxicity were also identified and included the heme oxygenase HMOX2, other detoxification enzymes and members of the ABC transporter family.

Chapter 4 reports identification of cellular mechanisms that modulate erythroid cell sensitivity to arsenic trioxide (ATO), a potent hematotoxicant and an effective anti-leukemic agent. A genome-wide loss-of-function CRISPR-based screen revealed novel molecular components influencing susceptibility of K562 erythroleukemic cells to ATO. Many of the candidates identified in the primary screen were simultaneously validated in a secondary screening approach. Functional enrichment analysis of validated genes revealed multiple pathways/processes implicated in the cellular response to ATO. The most significant pathway controls biosynthesis of selenocysteine, the 21st amino acid, and its incorporation into selenoproteins. Inactivation of components of this pathway resulted in notable cellular resistance to ATO. Based on the screening results, two models explaining the role of selenocysteine metabolism in ATO toxicity were proposed: the compromised thioredoxin reductase model and the arsenic-selenium-glutathione export model. Intriguingly, selenium pre-treatment of cells exhibited a protective effect against ATO cytotoxicity.

In chapter 5, susceptibility of erythroid cells to another hematotoxicant, acetaldehyde, was studied. Acetaldehyde, the primary product of alcohol metabolism and an endogenous metabolite, is a potential carcinogen and its well-established genotoxicity is thought to underlie bone marrow failure in Fanconi Anemia. A genome-wide knockout screen in erythroleukemic K562 cells identified several determinants of sensitivity to acetaldehyde. Consistent with the documented role of aldehydes in DNA damage, multiple identified candidate genes encode DNA repair enzymes. The top candidate gene encodes the tumor suppressor OVCA2, whose function is unknown. The uncovered role of OVCA2 in detoxifying acetaldehyde was validated in a secondary screen and by individual disruption of the OVCA2 gene. Interestingly, OVCA2 deficient cells displayed increased accumulation of the acetaldehyde-derived DNA adduct N2-ethylidene-2G.

Overall, these studies demonstrated the effectiveness of CRISPR/Cas9 loss-of-function genetic screening in deciphering mechanisms relevant to erythroid cell survival, proliferation, differentiation and response to injury.