Targeted Deletion of BCL11A Gene by CRISPR/Cas9 System for Fetal Hemoglobin Reactivation

Background and Aim: Beta-thalassemia is caused by unusual structureor decreased production of β-chains. On the other hands, HbF inductionwould be a promising strategy to attenuate the symptoms of thalassemia.Using the CRISPR/Cas9 technology which is a versatile and powerfulediting tool we proposed to reactivate HbF in the adult stage. B celllymphoma 11A (BCL11A) is a key regulatory agent in HbF silencing, sosuppression of the BCL11A protein could represent a therapeutic targetfor hemoglobinopathies disease.Methods: We proposed that knockdown of BCL11A expression byerythroid enhancer disruption would result in the induction of gammaglobinexpression. We used the CRISPR/Cas9 system to generate clonesof HEK293 cells with a deletion of the 200bp BCL11A enhancer. Thecleavage activity of each sgRNA was examined by T7EI assay. Weintroduced the SpCas9 and a pair of sgRNA expressing vectors intoHEK293 cells by Amaxa 2b-Nucleofector kit. Clonal isolation wasperformed in order to isolate clonal cells containing homozygotedeletions through serial dilution. Targeted genomic deletion via CRISPR/Cas9 using pairs of sgRNAs was evaluated and recognized by genomicPCR and Sanger sequencing. To assess the expression reduction ofBCL11A gene, BCL11A mRNA levels were checked by RT-qPCR. Results: Genomic PCR and Sanger sequencing results validated targeteddeletion in transfected cells and RT-qPCR results showed down to a twofoldreduction in BCL11A expression in HEK293 cells.Conclusion: This study showed that these CRISPR/cas9 constructs bringabout targeted deletion and reduction in BCL11A mRNA level. The nextstep of this study is to examine these constructs in other erythroleukemiacell lines in order to the elimination of the inhibitory effect of BCL11Aand induction of fetal hemoglobin production.

کلیدواژه ها:

Beta-thalassemia ، Fetal hemoglobin ، γ-globin ، CRISPR/Cas9 ، Genome editing