Session: 112. Thalassemia and Globin Gene Regulation: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Sickle Cell Disease, Thalassemia, Hemoglobinopathies, Diseases
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.
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