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Cell Discov. 2019 Aug 20;5:41. doi: 10.1038/s41421-019-0109-7. eCollection 2019.

Targeted exon skipping with AAV-mediated split adenine base editors.

Author information

1
1Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.
2
2Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.
3
3Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.
4
4Carle Illinois College of Medicine, Champaign, IL 61820 USA.
5
5Cancer Center at Illinois, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA.

Abstract

Techniques for exclusion of exons from mature transcripts have been applied as gene therapies for treating many different diseases. Since exon skipping has been traditionally accomplished using technologies that have a transient effect, it is particularly important to develop new techniques that enable permanent exon skipping. We have recently shown that this can be accomplished using cytidine base editors for permanently disabling the splice acceptor of target exons. We now demonstrate the application of CRISPR-Cas9 adenine deaminase base editors to disrupt the conserved adenine within splice acceptor sites for programmable exon skipping. We also demonstrate that by altering the amino acid sequence of the linker between the adenosine deaminase domain and the Cas9-nickase or by coupling the adenine base editor with a uracil glycosylase inhibitor, the DNA editing efficiency and exon-skipping rates improve significantly. Finally, we developed a split base editor architecture compatible with adeno-associated viral packaging. Collectively, these results represent significant progress toward permanent in vivo exon skipping through base editing and, ultimately, a new modality of gene therapy for the treatment of genetic diseases.

KEYWORDS:

Biological techniques; Transcription

Conflict of interest statement

Conflict of interestJ.W., M.G., W.W. and P.P. are named inventors on patent applications related to gene editing and exon skipping with base editors. The remaining authors declare that they have no conflict of interest.

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