A Novel Genome-Scale CRISPR-Cas12a Screen Identifies Ptpa As a Fetal Hemoglobin Regulator Via BCL11A

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 γ-globin expression in adult erythroid cells.

Because Cas9 technology is limiting at the genome-wide scale, we created 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.

Our screens identified PTPA (protein phosphatase 2 phosphatase activator) as a top candidate gene. It is a known modulator of PP2A, 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 PTPA in HUDEP2 cells significantly increased γ-globin mRNA and protein expression. Using sgRNA-Cas9 ribonucleoprotein complex nucleofection, PTPA 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. PTPA depletion in HUDEP2 cells markedly reduced BCL11A mRNA and protein levels. Furthermore, BCL11A overexpression in PTPA gene-edited cells reduced γ-globin mRNA commensurate to parental control cells, thereby demonstrating it mediates γ-globin silencing predominantly via BCL11A. Altogether, our studies utilizing a novel Cas12a-based, genome-wide screen nominate a new pathway mediating γ-globin silencing via regulation of BCL11A.

Abdulmalik:Name of Organization/ Type of relationship Illexcor Therapeutics: Research Funding. Blobel:Nucleome Therapeutics: Membership on an entity's Board of Directors or advisory committees; Design Therapeutics: Consultancy; Harmonic Discovery: Consultancy.