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ABSTRACT: Directed Evolution of Adenine Base Editors with Increased Activity and Therapeutic Application

PROVIDER: PRJNA574182 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10185769.fastq.gz	Fastqsanger.gz
	SRR11297700.fastq.gz	Fastqsanger.gz
	SRR11297800.fastq.gz	Fastqsanger.gz
	SRR11297900.fastq.gz	Fastqsanger.gz
	SRR11298000.fastq.gz	Fastqsanger.gz

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Directed Evolution of an Adenine Base Editor with Increased Activity and Context Compatibility

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Project description:Adenine base editors (ABEs) are precise gene-editing agents that convert A:T pairs into G:C through a deoxyinosine intermediate. ABEs function most effectively when the target A is in a TA context. Deficient ABE processing of RA (R = A or G) is most evident when the target A is outside the comfortable editing window or when delivery is suboptimal. In the current study, we report directed evolution of TadA8r, a new variant of the Escherichia coli tRNA-specific adenosine deaminase (TadA) with ultra-fast deoxyadenosine deamination and no context bias.

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Project description: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 demonstrated that this can be accomplished using cytidine base editors for permanently disabling the splice acceptor of target exons. We now demonstrate the application of adenine-deaminase base editors to disrupt the conserved adenosine 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 towards permanent in vivo exon skipping through base editing and, ultimately, a new modality of gene therapy for the treatment of genetic diseases.

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