@article {6585,
	title = {Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression.},
	journal = {Mol Ther},
	volume = {32},
	year = {2024},
	month = {2024 Mar 06},
	pages = {663-677},
	abstract = {<p>BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for β-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (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 used 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 elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in\&nbsp;vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in\&nbsp;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 effects. 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 because 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 that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.</p>
},
	keywords = {Fetal Hemoglobin, gamma-Globins, Gene Editing, Hematopoietic Stem Cells, Humans, Repressor Proteins, Zinc Fingers},
	issn = {1525-0024},
	doi = {10.1016/j.ymthe.2024.01.023},
	author = {Rajendiran, Vignesh and Devaraju, Nivedhitha and Haddad, Mahdi and Ravi, Nithin Sam and Panigrahi, Lokesh and Paul, Joshua and Gopalakrishnan, Chandrasekar and Wyman, Stacia and Ariudainambi, Keerthiga and Mahalingam, Gokulnath and Periyasami, Yogapriya and Prasad, Kirti and George, Anila and Sukumaran, Dhiyaneshwaran and Gopinathan, Sandhiya and Pai, Aswin Anand and Nakamura, Yukio and Balasubramanian, Poonkuzhali and Ramalingam, Rajasekaran and Thangavel, Saravanabhavan and Velayudhan, Shaji R and Corn, Jacon E and Mackay, Joel P and Marepally, Srujan and Srivastava, Alok and Crossley, Merlin and Mohankumar, Kumarasamypet M}
}
@article {2465,
	title = {Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin.},
	journal = {Elife},
	volume = {11},
	year = {2022},
	month = {2022 02 11},
	abstract = {<p>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 \&gt; C and -124T \&gt; 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.</p>
},
	keywords = {Adenine, Anemia, Sickle Cell, beta-Globins, beta-Thalassemia, Cell Line, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, Cytosine, Fetal Hemoglobin, gamma-Globins, Gene Editing, Hematopoietic Stem Cells, Humans, Point Mutation, Promoter Regions, Genetic},
	issn = {2050-084X},
	doi = {10.7554/eLife.65421},
	author = {Ravi, Nithin Sam and Wienert, Beeke and Wyman, Stacia K and Bell, Henry William and George, Anila and Mahalingam, Gokulnath and Vu, Jonathan T and Prasad, Kirti and Bandlamudi, Bhanu Prasad and Devaraju, Nivedhitha and Rajendiran, Vignesh and Syedbasha, Nazar and Pai, Aswin Anand and Nakamura, Yukio and Kurita, Ryo and Narayanasamy, Muthuraman and Balasubramanian, Poonkuzhali and Thangavel, Saravanabhavan and Marepally, Srujan and Velayudhan, Shaji R and Srivastava, Alok and DeWitt, Mark A and Crossley, Merlin and Corn, Jacob E and Mohankumar, Kumarasamypet M}
}
