An optimized CRISPR-Cas12a genome-wide screen reveals PTPA phosphatase pathway in fetal hemoglobin silencing

Reactivating the fetal globin genes HBG1 and HBG2 in adult erythroid cells represents a validated therapeutic approach for hemoglobinopathies. Central mediators of the fetal-to-adult hemoglobin transition include the direct transcriptional HBG1/2 repressors BCL11A, LRF, and NFIA/X. Limited-scale screens have expanded the regulatory circuity surrounding fetal globin silencing, but systematic genome-wide dissection of such pathways is lacking. We used a 2-tiered genetic screening strategy, a novel CRISPR-Cas12a-based screening platform followed by a domain-focused CRISPR-Cas9 screen, to interrogate all known human protein-coding genes for their impact on HBG1/2 regulation and erythroid cellular fitness, generating a comprehensive resource for the field. Among the top new hits was protein phosphatase 2A (PP2A) phosphatase activator (PTPA), an activator of the serine-threonine phosphatase PP2A whose loss elevates HBG1/2 levels while preserving erythroid differentiation. Phenotypic rescue experiments revealed that PTPA silences HBG1/2 expression primarily by regulating BCL11A expression. To our knowledge, this study represents the most comprehensive CRISPR dissection of HBG regulation to date, highlighting the power of Cas12a-based genome-scale screening for uncovering disease-relevant pathways.