Fluorescent Tagging of Endogenous Beta-Globin Gene in S-HUDEP2 Cell Model for Live Cell Monitoring of Beta-Globin Expression and Regulation

Abstract

Introduction: The translation of β-globin (HBB) gene is crucial for adult hemoglobin (HbA) formation, and naturally occurring mutations in HBB cause disorders of erythropoiesis. Therefore, understanding the mechanisms governing HBB regulation and expression has been an active area of research. We aim to develop a cell model to monitor HBB expression in live cells as a tool to gain better understanding of HBB expression, regulation and for the discovery of novel therapeutic targets. Methods: Sickle Human Umbilical Cord Derived Erythroid Progenitor cells (S-HUDEP2) are immortalized CD34+ hematopoietic stem and progenitor cells with homozygous sickle mutation that exclusively produce sickle hemoglobin upon erythroid differentiation. To generate the cell model in S-HUDEP2, we delivered HiFi SpCas9/gRNA targeting the C-terminus of HBB together with a double-stranded DNA donor template encoding self-cleaving peptide, eGFP, and β-globin polyA signal flanking homology arms for simultaneous expression of β-globin and eGFP under the HBB promoter. We created this clonal cell model and confirmed biallelic precise in-frame targeted integration by PCR, Sanger sequencing and digital droplet PCR copy number assay to ensure simultaneous monitoring of both HBB alleles. Results: To validate this model, we delivered HiFi SpCas9/R-66S sgRNA as ribonucleoprotein (RNP) targeting HBB currently in pre-clinical studies for sickle cell anemia (SCA). With RNP delivery, CRISPR/Cas9 double-strand breaks (DSBs) will lead to insertions and deletions (indels); we hypothesized that varying indel frequencies and patterns will result in change in GFP expression. After RNP delivery, we conducted 7 days of in vitro cellular expansion to allow for completion of editing and stabilization of GFP signal followed by 5 days of in vitro cellular differentiation. We identified 3 distinct populations based on GFP expression: GFP-, GFPlow, and GFPhigh. These cells were bulk sorted, genomic DNA was extracted and we analyzed indel patterns for each population through targeted amplicon Next Generation Sequencing (NGS). Each population had a unique indel pattern: confirming that GFP expression is based on genotype. In addition, an increase in GFP mean fluorescent intensity (MFI) was seen after in vitro differentiation with preservation of these distinct groups; indicative of expected increase in HBB transcription. Based on these observations, through use of ddPCR copy number assay, we determined that the creation of CRISPR/Cas9 mediated large deletion (>200bp) is the underlying mechanism leading to GFP loss seen in GFP- andGFPlow populations. Nonsense mutations occurring early in exon 1 of HBB resulting in premature translation-termination codons are resistant to nonsense mediated mRNA decay (NMD), a well-known cellular surveillance mechanism. We hypothesized that RNP induced small frameshift indels in exon 1 allow for NMD escape thus preserving HBB translation and GFP expression while large deletions do not, leading to GFP loss. To further validate this model and define its potential for broad applicability, we confirmed its ability to undergo normal differentiation based on erythroid specific cell markers when compared to untagged S-HUDEP2. We also confirmed our model's capacity to form Hb tetramer with α-chains using Hb native PAGE which allows for identification of intact Hb tetramers based on reference migration. Conclusion: We have successfully established and validated a clonal cell model with GFP tagged β-globin, which can be used to monitor HBB expression in live cells via GFP expression, eliminating the need for cellular fixation and HbA antibody staining. Our model may provide a useful tool for a broad range of applications, including the examination of cellular mechanisms underlying HBB expression and the screening of potential therapeutic targets for hemoglobinopathies and other disorders of erythropoiesis.