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. 2023 Nov;13(4):799-805.
doi: 10.34172/apb.2023.074. Epub 2023 Feb 21.

CRISPR/Cas9 Ablated BCL11A Unveils the Genes with Possible Role of Globin Switching

Fatemeh Movahedi Motlagh  1 , Hamid Reza Soleimanpour-Lichaei  2 , Mehdi Shamsara  3 , Azadeh Etemadzadeh  1 , Mohammad Hossein Modarressi  1
Affiliations

CRISPR/Cas9 Ablated BCL11A Unveils the Genes with Possible Role of Globin Switching

Fatemeh Movahedi Motlagh et al. Adv Pharm Bull. 2023 Nov.

Abstract

Purpose: Fetal hemoglobin (HbF) upregulation is a mitigating factor in β-hemoglobinopathies therapy like β-thalassemia and sickle cell diseases. Finding molecular mechanisms and the key regulators responsible for globin switching could be helpful to develop effective ways to HbF upregulation. In our prior in silico report, we identified a few factors that are likely to be responsible for globin switching. The goal of this study is to experimentally validate the factors.

Methods: We established K562 cell line with BCL11A knock down leading to increase in HBG1/2 using CRISPR/Cas9 system. Then, using quantitative polymerase chain reaction (qPCR), we determined the expression level of the factors which were previously identified in our prior in silico study.

Results: our analysis showed that BCL11A was substantially knocked down, resulting in the upregulation of HBG1/2 in the BCL11A-ablated K562 cells using CRISPR/Cas9 system. Additionally, the experimental data acquired in this study validated our prior bioinformatics findings about three potentially responsible genes for globin switching, namely HIST1H2Bl, TRIM58, and Al133243.2.

Conclusion: BCL11A is a promising candidate for the treatment of β-hemoglobinopathies, with high HbF reactivation. In addition, HIST1H2BL, TRIM58 and Al133243.2 are likely to be involved in the mechanism of hemoglobin switching. To further validate the selected genes, more experimental in vivo and in vitro studies are required.

Keywords: BCL11A knockdown; Beta hemoglobinopathies; CRISPR/Cas9; Fetal hemoglobin; Globin switching.

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Conflict of interest statement

The authors have no conflict of interest.

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