Mus musculus strain:C57BL6J
Ontology highlight
ABSTRACT:
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| DRR140994_1.fastq.gz | Fastqsanger.gz | |||
| DRR140994_2.fastq.gz | Fastqsanger.gz | |||
| DRR140995_1.fastq.gz | Fastqsanger.gz | |||
| DRR140995_2.fastq.gz | Fastqsanger.gz | |||
| DRR140996_1.fastq.gz | Fastqsanger.gz |
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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.
2021-07-14 | GSE160256 | GEO
Project description:Eliminating genome-wide and transcriptome-wide off-target mutations by base editor
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Project description:CRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells. Here we show that a CBE with rAPOBEC1 can cause extensive transcriptome-wide RNA cytosine deamination in human cells, inducing tens of thousands of C-to-uracil (U) edits with frequencies ranging from 0.07% to 100% in 38% - 58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, 5’ UTR, and 3’ UTR mutations. We engineered two CBE variants bearing rAPOBEC1 mutations that substantially decrease the numbers of RNA edits (reductions of >390-fold and >3,800-fold) in human cells. These variants also showed more precise on-target DNA editing and, with the majority of gRNAs tested, editing efficiencies comparable to those observed with wild-type CBE. Finally, we show that recently described adenine base editors (ABEs) can also induce transcriptome-wide RNA edits. These results have important implications for the research and therapeutic uses of base editors, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms. This SuperSeries is composed of the SubSeries listed below.
2019-03-31 | GSE121668 | GEO
Project description:CRISPR-Cas base editor technology enables targeted nucleotide alterations and is being rapidly deployed for research and potential therapeutic applications. The most widely used base editors induce DNA cytosine (C) deamination with rat APOBEC1 (rAPOBEC1) enzyme, which is targeted by a linked Cas protein-guide RNA (gRNA) complex. Previous studies of cytosine base editor (CBE) specificity have identified off-target DNA edits in human cells. Here we show that a CBE with rAPOBEC1 can cause extensive transcriptome-wide RNA cytosine deamination in human cells, inducing tens of thousands of C-to-uracil (U) edits with frequencies ranging from 0.07% to 100% in 38% - 58% of expressed genes. CBE-induced RNA edits occur in both protein-coding and non-protein-coding sequences and generate missense, nonsense, splice site, 5’ UTR, and 3’ UTR mutations. We engineered two CBE variants bearing rAPOBEC1 mutations that substantially decrease the numbers of RNA edits (reductions of >390-fold and >3,800-fold) in human cells. These variants also showed more precise on-target DNA editing and, with the majority of gRNAs tested, editing efficiencies comparable to those observed with wild-type CBE. Finally, we show that recently described adenine base editors (ABEs) can also induce transcriptome-wide RNA edits. These results have important implications for the research and therapeutic uses of base editors, illustrate the feasibility of engineering improved variants with reduced RNA editing activities, and suggest the need to more fully define and characterize the RNA off-target effects of deaminase enzymes in base editor platforms.
2019-03-31 | GSE126502 | GEO
Project description:Eliminating genome-wide and transcriptome-wide off-target mutations by base editor [tBE_target_genomic_seq]
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Project description:To investigate gene expression changes related to two fAD mutations (A79V and L150P) in the Presenilin-1 gene (PSEN1) we compared the transcriptomes (polyA and total) of glutamatergic cortical neurons derived from fAD-mutant human induced pluripotent stem cells and their individual isogenic controls generated via precision CRISPR/Cas9 genome editing.
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Project description:Current base editors use DNA deaminases, including cytidine deaminase in cytidine base editor (CBE) or adenine deaminase in adenine base editor (ABE), to facilitate transition nucleotide substitutions. Combining CBE or ABE with glycosylase enzymes can induce limited transversion mutations. Nonetheless, a critical demand remains for base editors capable of generating alternative mutation types, such as T>G corrections. In this study, we leveraged pre-trained protein language models to optimize a uracil-N-glycosylase (UNG) variant with altered specificity for thymines (eTDG). Notably, after two rounds of testing fewer than 50 top-ranking variants, more than 50% exhibited over 1.5-fold enhancement in enzymatic activities. When eTDG was fused with nCas9, it induced programmable T-to-S (G/C) substitutions and corrected db/db diabetic mutation in mice (up to 55%). Our findings not only establish orthogonal strategies for developing novel base editors, but also demonstrate the capacities of protein language models for optimizing enzymes without extensive task-specific training data.
2024-01-23 | GSE253655 | 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