Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Systematic comparison and base-editing-mediated directed protein evolution and functional screening yield superior auxin-inducible degron technology

ABSTRACT: Biological mechanisms are inherently dynamic, requiring precise and rapid manipulations for effective characterization. Traditional genetic manipulations operate on long timescales, making them unsuitable for studying dynamic processes or characterizing essential genes, where chronic depletion can cause cell death. We compared five inducible protein degradation systems—dTAG, HaloPROTAC, IKZF3, and two auxin-inducible degrons (AID) using OsTIR1 and AtFB2—evaluating degradation efficiency, basal degradation, target recovery after ligand washout, and ligand impact. This analysis identified OsTIR1-based AID 2.0 as the most robust system. However, AID 2.0's higher degradation efficiency came with target-specific basal degradation and slower recovery rates. To address these limitations, we employed base-editing-mediated mutagenesis followed by several rounds of functional selection and screening. This directed protein evolution generated several gain-of-function OsTIR1 variants, including S210A, that significantly enhanced the overall degron efficiency. The resulting degron system, named AID 2.1, maintains effective target protein depletion with minimal basal degradation and faster recovery after ligand washout, enabling characterization and rescue experiments for essential genes. Our comparative assessment and directed evolution approach provide a reference dataset and improved degron technology for studying gene functions in dynamic biological contexts.

ORGANISM(S): Homo sapiens

PROVIDER: GSE300048 | GEO | 2025/06/24

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Systematic comparison and base-editing-mediated directed protein evolution and functional screening yield superior auxin-inducible degron technology [2]

Project description:Biological mechanisms are inherently dynamic, requiring precise and rapid manipulations for effective characterization. Traditional genetic manipulations operate on long timescales, making them unsuitable for studying dynamic processes or characterizing essential genes, where chronic depletion can cause cell death. We compared five inducible protein degradation systems—dTAG, HaloPROTAC, IKZF3, and two auxin-inducible degrons (AID) using OsTIR1 and AtFB2—evaluating degradation efficiency, basal degradation, target recovery after ligand washout, and ligand impact. This analysis identified OsTIR1-based AID 2.0 as the most robust system. However, AID 2.0's higher degradation efficiency came with target-specific basal degradation and slower recovery rates. To address these limitations, we employed base-editing-mediated mutagenesis followed by several rounds of functional selection and screening. This directed protein evolution generated several gain-of-function OsTIR1 variants, including S210A, that significantly enhanced the overall degron efficiency. The resulting degron system, named AID 2.1, maintains effective target protein depletion with minimal basal degradation and faster recovery after ligand washout, enabling characterization and rescue experiments for essential genes. Our comparative assessment and directed evolution approach provide a reference dataset and improved degron technology for studying gene functions in dynamic biological contexts.

2025-06-26 | GSE300297 | GEO

Systematic comparison and base-editing-mediated directed protein evolution and functional screening yield superior auxin-inducible degron technology [1]

2025-06-26 | GSE300296 | GEO

RNA-seq on ES cells where DDX6 was knocked down by the AID method

Project description:To degrade DDX6 acutely, we adopted an advanced protein degradation method, the auxin-induced degron (AID) system. For this purpose, we prepared an AID-tagged DDX6 and E3 ligase subunit of OsTIR1 in an engineered ES (D6AdP cell line). These deposited data are RNA-seq data on these D6AdP ES cells treated with the auxin analog (IAA) treatment for 0, 3, and 6 hours.

2022-11-01 | GSE200483 | GEO

One-step generation of auxin-inducible degron cells with high-efficiency homozygous tagging

Project description:Auxin-inducible degron (AID) technology is powerful for chemogenetic control of proteolysis. However, generation of human cell lines to deplete endogenous proteins with AID remains challenging. Typically, homozygous degron-tagging efficiency is low and overexpression of an auxin receptor requires additional engineering steps. Here, we establish a one-step genome editing procedure with high-efficiency homozygous tagging and auxin receptor expression. We demonstrate its application in 5 human cell lines, including embryonic stem (ES) cells. The method allowed isolation of AID single-cell clones in 10 days for 11 target proteins with >80% average homozygous degron-tagging efficiency in A431 cells, and >50% efficiency for 5 targets in H9 ES cells. The tagged endogenous proteins were inducibly degraded in all cell lines, including ES cells and ES-cell derived neurons, with robust expected functional readouts. This method facilitates the application of AID for studying endogenous protein functions in human cells, especially in stem cells.

2024-01-31 | GSE243637 | GEO

mRNA-Seq of primary MEFs untreated or treated with auxin for OGT degradation

Project description:Primary mouse embryonic fibroblasts (MEFs) were derived from E12.5 embryos from the breeding of male mice which are homozygous for the insertion of the OsTIR1 transgene and females which are heterozygous for the AID knock-in at the endogenous Ogt locus (OgtAID/WT). The resulting MEFs clones were genotyped for sex and presence of the insertions and three pairs of OsTIR1-het;OgtAID/Y and control OsTIR1-het;OgtWT/Y male clones were used for the experiment, each pair coming from a different litter. The experiment consisted in treating each clone with auxin (IAA) for 24 hours, 48 hours or 96 hours and collecting them for single-end mRNA-Seq. One plate per clone was left untreated and also collected for mRNA-Seq.

2024-08-15 | E-MTAB-13298 | biostudies-arrayexpress

mRNA-seq analysis of Caenorhabditis elegans starved L1 larvae with and without AMA-1/Pol II degradation in the soma and germ line during L1 arrest

Project description:Whole starved L1 larvae were collected with and without AMA-1 degradation by the auxin-inducible degron (AID) system in the soma and germ line between 36 and 132 hours of starvation along with controls.

2022-10-11 | GSE173655 | GEO

Distinct properties and functions of CTCF revealed by a rapidly inducible degron system [PRO-Seq]

Project description:We coupled an auxin-induced degron (AID) system with precision nuclear run-on (PRO-seq). Oriented CTCF motifs in gene bodies are associated with transcriptional stalling in a manner ostensibly independent of bound CTCF. CTCF binding sites display highly variable resistance to degradation, with persistent sites displaying architectural related features.

2021-02-23 | GSE150414 | GEO

Distinct properties and functions of CTCF revealed by a rapidly inducible degron system [ChIP-Seq]

2021-02-23 | GSE150415 | GEO

Genome-Wide Transcriptional Effects of the Anti-Cancer Agent Camptothecin

Project description:The anti-cancer drug camptothecin inhibits replication and transcription by trapping DNA topoisomerase I (Top1) covalently to DNA in a M-bM-^@M-^\cleavable complexM-bM-^@M-^]. To examine the effects of camptothecin on RNA synthesis genome-wide we used Bru-Seq and show that camptothecin treatment affected transcription initiation, elongation, termination, splicing and enhancer activity. Following removal of camptothecin, transcription spread as a wave from the 5M-bM-^@M-^Y-end of genes with no recovery of transcription apparent from RNA polymerases stalled in the body of genes. As a result, camptothecin preferentially inhibited the expression of large genes such as proto-oncogenes, and anti-apoptotic genes while smaller ribosomal protein genes, pro-apoptotic genes and p53 target genes showed relative higher expression. In addition, a set of mitotic regulator genes and histone genes were inhibited in a size-independent manner. Cockayne syndrome group B fibroblasts showed a very similar RNA synthesis recovery profile to normal fibroblasts suggesting that transcription-coupled repair is not involved in the repair of transcription-blocking TOP1 lesions. These findings of the effects of camptothecin on transcription have important implications for its anti-cancer activities and may aid in the design of improved combinatorial treatments involving Top1 poisons. Analysis of the effect of Camptothecin (CPT) on transcription. Normal fibroblasts and Cockayne syndrome group B fibroblasts were exposed to CPT for 60 minutes, after which a washout was performed. Nascent RNA was labeled using bromouridine for 15 minutes starting at time points: 1) 15 minutes before the washout; 2) Immediately after the washout; 3) 15 minutes after the washout. Test samples are compared to control cells that were not exposed to CPT.

2013-10-29 | E-GEOD-48678 | biostudies-arrayexpress

Nucleosome position mapping by micrococcal nuclease analysis in S. cerevisiae of Top1-AID/Top2-AID strains with auxin in galactose-rich media

Project description:We analyed the nucleosome positions by using 2 concentrations of micrococcal nuclease of haploid yeast strains that were grown in galactose containing synthetic complete media. Strains contained AID-tags at the endogeous TOP1 and TOP2 genes. One strain contained OsTIR1 and these cells were either untreated or treated for 60 min with 500 uM auxin.

2023-04-28 | GSE196945 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data