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ABSTRACT: Engineering of efficient photoactivatable A to I RNA base editor for gene therapy in vivo

PROVIDER: PRJNA1194119 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR31644902_1.fastq.gz	Fastqsanger.gz
	SRR31644902_2.fastq.gz	Fastqsanger.gz
	SRR31644903_1.fastq.gz	Fastqsanger.gz
	SRR31644903_2.fastq.gz	Fastqsanger.gz
	SRR31644904_1.fastq.gz	Fastqsanger.gz

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Project description:Engineering of efficient photoactivatable A to I RNA base editor for gene therapy in vivo

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Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing

Project description:Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows for robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.

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Base editing of general transcription factor Spt15 to enhance yeast stress tolerance

Project description:We aimed to explore the application of the Target-AID base editor in genomic in situ protein engineering by generating nonsynonymous mutations. A general transcription factor Spt15 (TATA-box binding protein) gene of Saccharomyces cerevisiae was selected as a target. Based on computational and experimental scanning mutagenesis of the Spt15 gene as well as flask-fermentation screening, three stress-tolerant Spt15 mutant strains (A140G, P169A and R238K) and two stress-sensitive Spt15 mutant strains (S118L and L214V) were obtained. To validate the regulatory mechanisms underlying these different Spt15 mutants, genome-wide transcriptome analysis by RNA sequencing was carried out to quantify global transcription changes in the Spt15 mutant strains compared to the wild type strain at the same culture conditions including the unstressed normal condition as well as hyperosmotic and thermal stress conditions. Results uncover the impacts of the Spt15 point mutations on global transcriptional regulation in response to hyperosmotic and thermal stresses, and provide insight into the applicability of the Target-AID base editor in genomic in situ protein engineering to alter yeast stress tolerance.

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Developing a T>A Base Editor via Protein Universe Mining and optimizing translesion polymerase

Project description:A engineering Pol η variants offers the exciting prospect of further tailoring its activity for base editing. Improving its processivity could ensure that Pol η completes the bypass of the AP site and perhaps extends a few more bases before dissociating, thus preventing the recruitment of other, potentially less desirable, cellular polymerases or repair factors. Base on this hypothesis, We decide to develop a novel T-to-A base editor.

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

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.

2019-09-12 | GSE120125 | GEO

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