-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

1101 Genome-Scale CRISPR-Cas12a Screen Identifies Novel Fetal Hemoglobin Regulators

Program: Oral and Poster Abstracts
Session: 112. Thalassemia and Globin Gene Regulation: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Sickle Cell Disease, Thalassemia, Hemoglobinopathies, Diseases
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Elizabeth A. Traxler, MD, PhD1,2, Chad Komar3*, Megan Saari2*, A Josephine Thrasher4*, Kunhua Qin, PhD2*, Eugene Khandros, MD, PhD2, Quynn Hotan4*, Osheiza Abdulmalik, DVM2*, Claire Shao5*, Qingzhou Chen, PhD6*, Andy J. Minn, MD, PhD7*, Cheryl A. Keller, PhD8*, Junwei Shi, PhD9* and Gerd A. Blobel, MD, PhD2

1Division of Hematology/Oncology, Department of Medicine, Hospital of University of Pennsylvania, Philadelphia, PA
2Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA
3Perelman School of Medicine at University of Pennsylvania, Philadelphia
4Division of Hematology, Children's Hospital of Philadelphia, Philadelphia
5Department of Radiation Oncology, University of Pennsylvania, Philadelphia
6Department of Cancer Biology, Perelman School of Medicine at University of Pennsylvania, Philadelphia
7Department of Radiation Oncology, Perelman School of Medicine at University of Pennsylvania, Philadelphia, PA
8Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA
9Department of Cancer Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA

Reversal of the postnatal hemoglobin switch in red blood cells is a promising approach to therapies for sickle cell disease and β-thalassemia, as reactivating fetal hemoglobin (HbF, α2γ2) in adult red blood cells ameliorates disease complications. Studies over several decades have illuminated the paradigm of this fetal-to-adult hemoglobin transition, including central roles of transcriptional repressors BCL11A, LRF, and NFIA/X, which recruit repressive chromatin modifiers to the γ-globin locus. To elucidate undescribed regulatory mechanisms of these molecules and reveal new targets for therapies, we performed the first genome-wide genetic screen to pinpoint specific pathways or protein complexes that regulate HbF expression in adult erythroid cells.

Because Cas9 technology is limiting at the genome-wide scale, we conducted a CRISPR-Cas12a genetic screen targeting all known coding genes in the human genome using a novel custom array directed towards 18,292 genes. The single CRISPR RNA (crRNA) library was cloned into a lentivirus scaffold and transduced into the adult-type erythroid cell line, HUDEP2, engineered to stably express Cas12a. HUDEP2 cells express little HbF, allowing for positive phenotypic selection (HbF+) by fluorescence-activated cell-sorting. By isolating the top 10% and bottom 10% of HbF-expressing cells and determining the representation of crRNAs via deep sequencing, we identified a crRNA subset enriched in the high-HbF population encompassing 211 candidate genes, representing potential regulators of HbF. To prioritize these genes for follow up studies, we conducted a domain-focused, CRISPR-Cas9 screen of all 211 candidate genes. Using 2,184 sgRNAs, this validation Cas9 screen in HUDEP2 cells nominated 18 high-confidence targets.

Two of the top candidate genes were identified as modulators of the phosphatase PP2A, PPP2R4 and TIPRL. PP2A is a multi-subunit complex with diverse cellular functions as a serine/threonine phosphatase. To our knowledge, it has no previously known role on globin gene production. In validation experiments, disruption of either PPP2R4 or TIPRL in HUDEP2 cells significantly increased γ-globin mRNA and protein expression. Using sgRNA-Cas9 ribonucleoprotein complex nucleofection, PPP2R4 gene editing in primary human CD34+ cell derived erythroblasts significantly increased γ-globin mRNA and protein expression without overtly affecting erythroid maturation. Notably, the degree of effect is similar to that of BCL11A +58 enhancer disruption. Depletion of either PP2A regulator in HUDEP2 cells resulted in markedly reduced BCL11A protein levels. Furthermore, BCL11A overexpression in PPP2R4 or TIPRL-targeted cells reduced γ-globin mRNA commensurate to parental control cells, thereby demonstrating both TIPRL and PPP2R4 mediate γ-globin silencing predominantly via BCL11A. RNA-sequencing of TIPRL and PPP2R4-edited HUDEP2 cells revealed strong overlap of differentially expressed genes, consistent with convergent function. Altogether, our studies utilizing a novel Cas12a-based, genome-wide screen nominate a new pathway mediating γ-globin silencing via regulation of BCL11A.

Disclosures: Minn: Merck: Research Funding; Takeda: Consultancy; Xilio: Consultancy; H3 Biomedicine and Related Sciences: Consultancy. Blobel: Blueprint Medicine: Research Funding; Fulcrum Therapeutics: Research Funding.

*signifies non-member of ASH