Editing of homologous globin genes by nickase-deficient base editor mitigates large intergenic deletions in HSPCs

Recent studies have shown that base editing, even with single-strand breaks, could result in large deletions of the interstitial regions while targeting homologous regions. Several therapeutically relevant genes such as HBG, HBB, CCR5, and CD33 have homologous sites and are prone for large deletion...

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Bibliographic Details
Published in:	Molecular therapy. Nucleic acids Vol. 35; no. 4; p. 102347
Main Authors:	George, Anila, Sadanandan, Poornasree, Ravi, Nithin Sam, Vaishnavi, B., Marepally, Srujan, Thangavel, Saravanbhavan, Velayudhan, Shaji R., Srivastava, Alok, Mohankumar, Kumarasamypet M
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 10-12-2024 American Society of Gene & Cell Therapy Elsevier
Subjects:	base editing beta globin Brief Report CRISPR-Cas9 gamma globin gene editing hemoglobinopathies large deletions MT: RNA/DNA Editing multiplexed editing MT: RNA/DNA Editing hemoglobinopathies large deletions CRISPR-Cas9 beta globin gamma globin multiplexed editing base editing gene editing
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Summary:	Recent studies have shown that base editing, even with single-strand breaks, could result in large deletions of the interstitial regions while targeting homologous regions. Several therapeutically relevant genes such as HBG, HBB, CCR5, and CD33 have homologous sites and are prone for large deletion with base editing. Although the deletion frequency and indels observed are lesser than what is obtained with Cas9, they could still diminish therapeutic efficacy. We sought to evaluate whether these deletions could be overcome while maintaining editing efficiency by using dCas9 fusion of ABE8e in the place of nickaseCas9. Using guide RNAs (gRNAs) targeting the γ-globin promoter and the β-globin exon, we evaluated the editing outcome and frequency of large deletion using nABE8e and dABE8e in human HSPCs. We show that dABE8e can edit efficiently while abolishing the formation of large interstitial deletions. Furthermore, this approach enabled efficient multiplexed base editing on complementary strands without generating insertions and deletions. Removal of nickase activity improves the precision of base editing, thus making it a safer approach for therapeutic genome editing. [Display omitted] Mohankumar and colleagues show that DNA nicks are the main cause for the formation of indels and large deletions while using base editors in homologous regions. They describe the use of a nickase-deficient base editor for efficient editing at these regions without inducing large deletions in human HSPCs.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	2162-2531 2162-2531
DOI:	10.1016/j.omtn.2024.102347