Ontology highlight
ABSTRACT:
PROVIDER: PRJNA746395 | ENA |
REPOSITORIES: ENA
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Project description:Genome-wide knockout or knockdown screens have become powerful tools for the investigation of genotype-to-phenotype relationships. In bacteria, these screens commonly rely on transcriptional repression by dCas9, gene knockouts through Cas9 editing or random transposon mutagenesis, but depending on the technique, suffer from incomplete gene silencing, low editing efficiencies or they require massive library sizes. Here, we take a distinct approach with base editing to introduce premature stop codons or mutate start codons in Escherichia coli using a ScCas9 nickase derived base editor (ScBE3) that exhibits flexible PAM recognition. We then derive guide design rules by applying machine learning to a gene essentiality screen conducted in E. coli. For further improvement, we combined base-editing with Cas9-induced cleavage of the unedited cell fraction. The efficiency of this dual system was validated through a screen of conditionally essential E. coli genes. This improved setup that decouples the gene editing from the screening leads to more efficient guide depletion and confirmed previously published conditionally essential genes. Overall, base editing represents a useful tool for genome-wide knockout screens in bacteria and will eventually enable genome-wide knockout screens in a broader range of bacterial species to study their diverse genetics.
2024-02-05 | GSE225335 | GEO
Project description:Background: RNA editing encompasses a post-transcriptional process in which the genomically templated sequence is enzymatically altered and introduces a modified base into the edited transcript. Mammalian C-to-U RNA editing represents a distinct subtype of base modification, whose prototype is intestinal apolipoproteinB (apoB) mRNA, mediated by the catalytic deaminase Apobec-1. However, the genome-wide identification, tissue-specificity and functional implications of Apobec-1 mediated C-to-U RNA editing remains incomplete. Results: Deep sequencing, data filtering and Sanger-sequence validation of intestinal and hepatic RNA from wild-type and Apobec-1 deficient mice revealed 56 novel editing sites in 54 intestinal mRNAs and 22 novel sites in 17 liver mRNAs (74-81% Sanger sequenced validated), all within 3’ untranslated regions. Eleven of 17 liver RNAs shared editing sites with intestinal RNAs, while 6 sites were unique to liver. Changes in RNA editing led to corresponding changes in intestinal mRNA and protein levels in 11 genes. RNA editing in vivo following tissue-specific Apobec-1 adenoviral or transgenic Apobec-1 overexpression revealed that a subset of targets identified in wild-type mice were restored in Apobec-1 deficient mouse intestine and liver following Apobec-1 rescue. We found distinctive polysome profiles for several RNA editing targets and demonstrated novel exonic editing sites in nuclear preparations from intestine (but not hepatic) apoB RNA. RNA editing was validated using cell-free extracts from wild-type but not Apobec-1 deficient mice, demonstrating that Apobec-1 is required. Conclusions: These studies define selective, tissue-specific targets of Apobec-1 dependent RNA editing and show the functional consequences of editing are both transcript- and tissue-specific.
2014-06-02 | PXD001007 | Pride
Project description:Even the latest generation of base editor (BE3) causes unwanted indels and non-C-to-T substitutions, compromising the fidelity of base editing outcome. Here we report a enhanced base editing system. The enhanced base edting system decreased the formation of unintended indels and C-to-A/C-to-G substitutions, and increased the frequency of desired C-to-T substitution, thereby improving both the accuracy and efficiency of base editing.
2017-08-29 | GSE98685 | GEO
Project description:Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow rich phenotyping through single-cell RNA sequencing readouts, and separately, characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.
2023-05-02 | GSE215253 | GEO