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Developing a T>A Base Editor via Protein Universe Mining and optimizing translesion polymerase

ABSTRACT: Developing a T>A Base Editor via Protein Universe Mining and optimizing translesion polymerase

PROVIDER: PRJNA1284345 | ENA |

REPOSITORIES: ENA

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Developing a T>A Base Editor via Protein Universe Mining and optimizing translesion polymerase

Project description:A engineering Pol η variants offers the exciting prospect of further tailoring its activity for base editing. Improving its processivity could ensure that Pol η completes the bypass of the AP site and perhaps extends a few more bases before dissociating, thus preventing the recruitment of other, potentially less desirable, cellular polymerases or repair factors. Base on this hypothesis, We decide to develop a novel T-to-A base editor.

2025-07-08 | GSE301258 | GEO

Genome-wide base editor screen identifies regulators of protein abundance in yeast

Project description:Proteins are key molecular players in a cell, and their abundance is extensively regulated not just at the level of gene expression but also post-transcriptionally. Here, we describe a genetic screen in yeast that enables systematic characterization of how protein abundance regulation is encoded in the genome. The screen combines a CRISPR/Cas9 base editor to introduce point mutations with fluorescent tagging of endogenous proteins to facilitate a flow-cytometric readout. We first benchmarked base editor performance in yeast with individual gRNAs as well as in positive and negative selection screens. We then examined the effects of 16,452 genetic perturbations on the abundance of eleven proteins representing a variety of cellular functions. We uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Overall, our novel screening approach provides unprecedented insights into the components, scale and connectedness of the protein regulatory network.

2022-11-07 | PXD029363 | Pride

Developing a highly efficient CGBE base editor in watermelon.

Project description:Cytosine and adenosine base editors (CBEs and ABEs) are novel genome-editing tools that have been widely utilized in molecular breeding to precisely modify single-nucleotide polymorphisms (SNPs) critical for plant agronomic traits and species evolution. However, conventional BE editors are limited to achieve C-to-T and A-to-G substitutions, respectively. To enhance the applicability of base editing technology in watermelon, we developed an efficient CGBE editor (SCGBE2.0) by removing the uracil glycosylase inhibitor (UGI) unit from the commonly used hA3A-CBE and incorporating the uracil-DNA glycosylase (UNG) component. Seven specific guide RNAs (sgRNAs) targeting five watermelon genes were designed to assess the editing efficiency of SCGBE. The results obtained from stably transformed watermelon plants demonstrated that SCGBE2.0 could efficiently induce C-to-G mutations at positions C5-C9 in 43.2% transgenic plants (with a maximum base conversion efficiency of 46.1%) and C-to-A mutation at position C4 in 23.5% transgenic plants (with a maximum base conversion efficiency of 45.9%). These findings highlight the capability of our integrated SCGBE2.0 editor to achieve C-to-G/A mutations in a site-preferred manner, thus providing an efficient base editing tool for precise base modification and site-directed saturated mutagenesis in watermelon.

| S-EPMC10500149 | biostudies-literature

Optimizing plant adenine base editor systems by modifying the transgene selection system.

Project description: Not available

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Determinants of base editor efficacy are position-specific

Project description:Determinants of base editor efficacy are position-specific

| PRJEB44304 | ENA

Developing & Optimizing Acute Myeloid Leukemia Specific Therapeutic Antibodies

Project description:Developing & Optimizing Acute Myeloid Leukemia Specific Therapeutic Antibodies

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Transcriptome wide edits after split-engineered base editor treatment

Project description:Transcriptome wide edits after split-engineered base editor treatment

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Multiplexed inactivation of endogenous genes by base-editor-mediated multi-stop system

Project description:Base editor is also a powerful tool to introduce early stop codons to simultaneously silence multiple genes to study family gene function in embryo development. Here, we identified the highest efficiency base editing system (hA3A-eBE-Y130F) with few sgRNA independent off-target sites among six well-established base editors in mouse embryo. Further, we developed a Multi-Stop system to simultaneously knock-out all Ten-eleven translocation (TET) family of dioxygenase genes (Tet1, Tet2 and Tet3) in zygotic genome leads embryo die before 10.5 days of gestation. Moreover, we showed that inactivation of three DNA methyltransferases genes (Dnmt1, Dnmt3a and Dnmt3b) in zygote leads to gastrulation failure. We also obtained multiple TET and DNMT family genes (6KO: Dnmt1/3a/3b and Tet1/2/3; 5KO: Dnmt3a/3b and Tet1/2/3; 4KO: Dnmt1 and Tet1/2/3) deactivated mouse embryos in one step, which indicated that Dnmt1 or Dnmt3a/b is indispensable for mouse gastrulation.

2023-04-19 | GSE188658 | GEO

Base-editor-mediated multiplexed inactivation of DNA methyltransferases reveals essential roles of miRNAs in mouse gastrulation

Project description:Here, we first establish an easy Multi-Stop system to simultaneously inactivate genes involved in DNA methylation and demethylation in zygotes through introduction of the stop codon by hA3A-eBE-Y130F-mediated base editor (BE). While Multi-Stop-derived Dnmt-null embryos display embryonic lethal due to gastrulation failure. Moreover, mutation combinations between Tet and Dnmt families show severe embryonic lethal and Dnmt1 or Dnmt3a/3b is indispensable for mouse gastrulation. Then WGBS and RNA-seq analysis of different mutant embryos reveals genes jointly maintained by Dnmt1 and Dnmt3a/3b that are critical for gastrulation.

2023-04-23 | GSE162903 | GEO

Developing & Optimizing Acute Myeloid Leukemia Specific Therapeutic Antibodies [CRISPR Screen]

Project description:Developing & Optimizing Acute Myeloid Leukemia Specific Therapeutic Antibodies [CRISPR Screen]

| PRJNA843099 | ENA