Ontology highlight
ABSTRACT:
PROVIDER: PRJNA616114 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11444700.fastq.gz | Fastqsanger.gz | |||
| SRR11444701.fastq.gz | Fastqsanger.gz | |||
| SRR11444702.fastq.gz | Fastqsanger.gz | |||
| SRR11444703.fastq.gz | Fastqsanger.gz | |||
| SRR11444704.fastq.gz | Fastqsanger.gz |
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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
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.
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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.
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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.
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Project description:The therapeutic application of base editors is currently limited by their large sizes, which are often beyond the packaging capabilities of adeno-associated viral (AAV) vectors. Despite recent progress in mega genome mining that has identified a diverse array of compact CRISPR proteins (e.g. Cas12f, TnpB, and IscB), the resulting miniature base editors are often exhibited reduced activities and a limited targeting scope, leaving a broad spectrum of disease-relevant genetic variants inaccessible. In this study, we have developed a platform, designated as Zinc Finger Proteins (ZFP)-enhanced miniature base editor (zmBE), which integrates programmable DNA-binding domains to enhance the efficiency and expand targeting scope of miniature base editors, including those based on Un1Cas12f1 and OgeuIscB. Utilizing protein language model (PLM) for the design of ZFPs further simplified development and optimization of zmBEs tailored to a specific target. Leveraging these methodologies, we engineered a zmBE that effectively induced the SMN2 exon 7 A6>G conversion and restored SMN2 exon 7 inclusion. Our study thus provides a versatile platform for developing miniature base editors for in vivo therapeutic applications.
2025-04-18 | GSE294498 | GEO
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
Project description:We used an adenine base editor to target the translation start site and mRNA splicing site of Camk2d in order to knock out CaMKIIδ. We found that editing the 5' splice site of intron 7 can lead to premature translation termination, effectively knocking out CaMKIIδ.
2025-01-21 | GSE266652 | GEO
Project description:We used an adenine base editor to target the translation start site and mRNA splicing site of Camk2d in order to knock out CaMKIIδ. We found that editing the 5' splice site of intron 7 can lead to premature translation termination, effectively knocking out CaMKIIδ.
2025-01-21 | GSE265935 | GEO
Project description:Adenine and cytosine base editors (ABEs and CBEs) represent a new genome editing technology that allows the programmable installation of A-to-G or C-to-T alterations on DNA. We engineered Streptococcus pyogenes Cas9-based adenine and cytosine base editor (SpACE) that enables efficient simultaneous introduction of A-to-G and C-to-T substitutions in the same base editing window on DNA.
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