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ABSTRACT: Expanding the genome-targeting scope and the site selectivity of high-precision base editors

PROVIDER: PRJNA562458 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10023886_1.fastq.gz	Fastqsanger.gz
	SRR10023886_2.fastq.gz	Fastqsanger.gz
	SRR10023887.fastq.gz	Fastqsanger.gz
	SRR10023887_1.fastq.gz	Fastqsanger.gz
	SRR10023887_2.fastq.gz	Fastqsanger.gz

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Saccharomyces cerevisiae

Project description:Engineering of high-precision C-to-G base editors with expanded site selectivity and target compatibility

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Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope [DNA-seq]

Project description:Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the selected genomic locus, and is limited to a window within the CRISPR-Cas R-loop where single stranded (ss)DNA is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility, and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with an adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABE) with PAM-proximally shifted editing windows. HNHx-ABEs are substantially reduced in size, and expand the targeting scope of base editors. Our finding that the HNH domain is replaceable could moreover benefit future protein engineering efforts, where Cas9 operates together with other enzyme domains.

2020-11-12 | GSE161380 | GEO

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope [RNA-seq]

2020-11-12 | GSE161379 | GEO

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

2020-11-12 | GSE161293 | GEO

One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids

Project description:Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show Efficacy of cytosine and adenine base editors in modelingCTNNB1hot-spot mutations in hepatocyte organoids. Next, we use C>T base editors to insert nonsense mutations inPTENin endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug screening assays on organoids harboring eitherPTENorPTENandPIK3CAmutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C>T and A>G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.

2023-07-30 | GSE236490 | GEO

Homo sapiens

Project description:Circularly permuted and modified-PAM base editors with diversified targeting scope

| PRJNA498804 | ENA

Project description:Engineering Nme2Cas9 Adenine Base Editors with Improved Activity and Targeting Scope

| PRJNA1033663 | ENA

Project description:Expanding the scope of base editing

| PRJNA359383 | ENA

Efficient C-to-T base editing in methylated region with human APOBEC3A

Project description:Base editors (BEs) shed new light on correcting disease-related T-to-C mutations. However, current rat APOBEC1-based BEs are less efficient in editing cytosines in highly-methylated regions or in GpC context. By screening a variety of APOBEC/AID deaminases, we showed that human APOBEC3A-conjugated BE and its engineered forms can mediate efficient C-to-T base editing in all examined contexts, including regions with high-methylation levels and GpC dinucleotides, which extends base editing scope.

2018-08-20 | GSE114999 | GEO

CRISPR-SKIP: Programmable Gene Splicing with Single Base Editors [RNA-seq]

Project description:CRISPR gene editing has revolutionized biomedicine and biotechnology by providing a simple means to engineer genes in vivo by introducing mutations at target sites in the genomic DNA of living cells. However, given the stochasticity of cellular DNA repair mechanisms and the potential for introducing mutations at off-target sites, technologies capable of introducing targeted changes with increased precision, such as cytidine deaminase single-base editors, are preferred. We here present a versatile method termed CRISPR-SKIP that utilizes cytidine deaminase single-base editors to program de-novo exon skipping by mutating target DNA bases within splice acceptor sites. Given its simplicity and precision, CRISPR-SKIP will be broadly applicable in gene therapy and synthetic biology.

2018-07-06 | GSE111643 | GEO

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