bBase editing of key residues in the BCL11A-XL-specific zinc finger domains de-represses fetal globin expression

BCL11A-XL directly binds and represses the γ-globin (HBG1/2) gene promoters, using three Zinc-Finger domains (ZnF4, ZnF5, ZnF6), and is a potential target for β-hemoglobinopathy treatments. Disrupting BCL11A-XL results in de-repression of fetal globin and high HbF, but also affects Hematopoietic Stem and Progenitor Cells (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we employed both CRISPR/Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR/Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and the elevated HbF levels but impacted normal hematopoiesis. Far fewer side effects were observed with base editing, for instance, erythroid maturation in vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B-cell development in vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different impacts. Disrupting ZnF4 elevated HbF levels significantly, while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected as this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL required to repress γ-globin expression and provides a framework for exploring the introduction of natural mutations that may enable de-repression of single genes while leaving other functions unaffected.