Ontology highlight
ABSTRACT:
PROVIDER: PRJNA475983 | ENA |
REPOSITORIES: ENA
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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 | GSE111645 | GEO
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 | GSE111644 | GEO
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
Project description:CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.
2019-07-11 | GSE129893 | GEO
Project description:CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.
2019-07-11 | GSE129889 | GEO
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
Project description:The advent of base editors (BEs) holds a promising potential in correcting pathogenic-related point mutations to treat relevant diseases. Unexpectedly, Cas9 nickase (nCas9) derived BEs lead to DNA double-strand breaks, which can trigger unwanted cellular responses including a p53-mediated DNA damage response (DDR). Here, we showed that catalytically-dead-Cas12a (dCas12a) conjugated BEs induced no DNA break and minimally activated DDR proteins including H2AX, ATM, ATR and p53. We further developed a BEACON (Base Editing induced by human APOBEC3A and Cas12a without DNA break) system that fuses dCas12a to the engineered APOBEC3A with enhanced deamination efficiency and editing specificity. By using BEACON, efficient C-to-T editing was achieved at levels comparable to AncBE4max and only low levels of DDR and RNA off-target (OT) effects were triggered in mammalian cells. BEACON also induced in vivo base editing in mouse embryos and targeted C-to-T conversions were detected in F0 mice.
2020-05-05 | GSE145550 | GEO
Project description:The advent of base editors (BEs) holds a promising potential in correcting pathogenic-related point mutations to treat relevant diseases. Unexpectedly, Cas9 nickase (nCas9) derived BEs lead to DNA double-strand breaks, which can trigger unwanted cellular responses including a p53-mediated DNA damage response (DDR). Here, we showed that catalytically-dead-Cas12a (dCas12a) conjugated BEs induced no DNA break and minimally activated DDR proteins including H2AX, ATM, ATR and p53. We further developed a BEACON (Base Editing induced by human APOBEC3A and Cas12a without DNA break) system that fuses dCas12a to the engineered APOBEC3A with enhanced deamination efficiency and editing specificity. By using BEACON, efficient C-to-T editing was achieved at levels comparable to AncBE4max and only low levels of DDR and RNA off-target (OT) effects were triggered in mammalian cells. BEACON also induced in vivo base editing in mouse embryos and targeted C-to-T conversions were detected in F0 mice.
2020-05-05 | GSE144278 | GEO
Project description:Recent optimization of CRISPR/Cas9-mediated genome engineering has resulted in the development of base editors that can efficiently mediate C>T and A>G transitions. Combining these genome engineering tools with human adult stem cell (ASC)-derived organoid technology holds promise for disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived hepatocyte, endometrial and intestinal organoids. First, using conventional and evolved Cas9-variants, we show efficacy of both cytosine and adenine base editors and use them to model four hot-spot point mutations in CTNNB1 in hepatocyte organoids. Next, we apply C>T base editors in endometrial organoids to insert nonsense mutations in PTEN and demonstrate tumorigenicity even in the heterozygous state. Furthermore, we use cytosine base editors for simultaneous oncogene activation (PIK3CA) and tumor-suppressor inactivation (APC and TP53). To increase the flexibility of base editor multiplexing, we then combine SpCas9 and SaCas9 base editors for simultaneous C>T and A>G editing at individual target sites. Finally, we show the power of base editor multiplexing by modeling colorectal tumorigenesis in a single step by simultaneously transfecting sgRNA’s targeting four cancer genes.
Seven clonal organoid lines and one bulk wild-type control sample were paired-end whole-genome sequenced using the Illumina Novaseq 6000 system. We sequenced four clonal intestinal organoid lines harbouring engineered TP53 and FBXW7 mutations as well as three lines targeted for oncogenic APC/TP53/PIK3CA/SMAD4 mutations. This WGS showed, as previously reported, a genome-wide increase in C>T mutations due to C>T base editor off-target activity, which is not enriched in predicted off-target regions based on the sgRNA sequences. Furthermore, we confirmed the absence of editing-induced driver mutations and lack of off-target mutational hotspots created by the genomic engineering.
| EGAS00001006886 | EGA
Project description:We have developed a therapeutic strategy for beta-hemoglobinopathies aimed at reactivating fetal hemoglobin expression in red blood cells derived from human hematopoietic stem/progenitor cells edited with CRISPR/Cas9 nucleases, cytidine or adenine base editors targeting the fetal gamma-globin promoters. Here, we report the transcriptomic changes occurring in human hematopoietic stem/progenitor cells (obtained from healthy donors) 48 h after transfection with CRISPR/Cas9 nucleases, cytidine or adenine base editors.
2022-09-06 | GSE191135 | GEO