High-resolution functional mapping of non-coding sequences regulating fetal hemoglobin - High-resolution functional mapping of non-coding sequences regulating fetal hemoglobin Transcriptional regulation from non-coding sequences and interacting DNA-binding transcription factors (TF) govern the developmental switch from fetal to adult hemoglobin, commonly referred to as “hemoglobin switching”. The reinduction of fetal hemoglobin (HbF) has been recognized as a promising therapeutic strategy for β-hemoglobinopathies such as sickle-cell disease and β-thalassemia. Therefore, characterizing the cis- regulatory logic of sequences regulating HBG1 and HBG2, the genes expressing the γ-globin chains of HbF, is critical for understanding hemoglobin switching and developing more effective therapies. While population genetics studies have identified functional single-nucleotide variants (SNVs) relevant for HbF induction, this approach is limited to natural variation preventing comprehensive understanding of critical regulatory elements. With the advent of CRISPR-Cas9 genome editing, targeted mutagenesis of regulatory sequences has been an effective strategy for observing the functional consequence of variants unobserved in the population. Previous studies have identified three erythroid-specific enhancers of BCL11A, an HbF repressor, and performed dense SpCas9 nuclease dense mutagenesis throughout each enhancer, ultimately identifying a guide RNA (gRNA) hit targeting a TF binding site causing potent re-induction of HbF, recently translated into an FDA-approved gene therapy. However, due to limitations regarding the restrictive protospacer-adjacent motif, non-precise editing via large insertions/deletions, and indirect readout of editing via guide RNA (gRNA) sequencing, the limited resolution of these initial and subsequent studies prevent the comprehensive identification of HbF regulatory elements. By directly addressing these limitations, I hypothesize that high-throughput, PAM-flexible dense base editing of HbF regulatory sequences can elucidate the variant-level cis-regulatory logic and corresponding transcription factor (TF) binding sites underpinning hemoglobin switching, therefore capturing biological insights missed to date. Specifically, I will perform in-situ dense base-editing mutagenesis of three known erythroid- specific BCL11A enhancers and the HBG promoter due to their therapeutic relevance for potent HbF reinduction. To determine the variant-level cis-regulatory logic, I will perform two approaches: (Aim 1) I will indirectly estimate per-variant enrichment scores by statistical modelling of gRNA enrichment and surrogate-editing patterns using a gRNA sensor construct containing a target-mimicking “surrogate” sequence immediately downstream of the gRNA, and (Aim 2) I will directly estimate per-variant enrichment scores by statistical modelling of the allele enrichment from direct amplicon-sequencing of the target region. This work can uncover new therapeutically relevant gRNAs for β-hemoglobinopathies and establish a general approach that can be applied to study sequences regulating other therapeutically relevant phenotypes.
