TY - JOUR T1 - Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin. JF - Elife Y1 - 2022 A1 - Ravi, Nithin Sam A1 - Wienert, Beeke A1 - Wyman, Stacia K A1 - Bell, Henry William A1 - George, Anila A1 - Mahalingam, Gokulnath A1 - Vu, Jonathan T A1 - Prasad, Kirti A1 - Bandlamudi, Bhanu Prasad A1 - Devaraju, Nivedhitha A1 - Rajendiran, Vignesh A1 - Syedbasha, Nazar A1 - Pai, Aswin Anand A1 - Nakamura, Yukio A1 - Kurita, Ryo A1 - Narayanasamy, Muthuraman A1 - Balasubramanian, Poonkuzhali A1 - Thangavel, Saravanabhavan A1 - Marepally, Srujan A1 - Velayudhan, Shaji R A1 - Srivastava, Alok A1 - DeWitt, Mark A A1 - Crossley, Merlin A1 - Corn, Jacob E A1 - Mohankumar, Kumarasamypet M KW - Adenine KW - Anemia, Sickle Cell KW - beta-Globins KW - beta-Thalassemia KW - Cell Line KW - Clustered Regularly Interspaced Short Palindromic Repeats KW - CRISPR-Cas Systems KW - Cytosine KW - Fetal Hemoglobin KW - gamma-Globins KW - Gene Editing KW - Hematopoietic Stem Cells KW - Humans KW - Point Mutation KW - Promoter Regions, Genetic AB -

Naturally occurring point mutations in the promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

VL - 11 ER -