Ontology highlight
ABSTRACT:
PROVIDER: PRJNA782178 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR16991300_1.fastq.gz | Fastqsanger.gz | |||
| SRR16991300_2.fastq.gz | Fastqsanger.gz | |||
| SRR16991301_1.fastq.gz | Fastqsanger.gz | |||
| SRR16991301_2.fastq.gz | Fastqsanger.gz | |||
| SRR16991302_1.fastq.gz | Fastqsanger.gz |
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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: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.
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| EGAS00001006886 | EGA