ArticleProceedings of the National Academy of Sciences of the United States of America2023
Efficient tagging of endogenous proteins in human cell lines for structural studies by single-particle cryo-EM.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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15 citing papers in PubMed, 10 citations in OpenAlex.
- HIT-MAP: A scalable approach to multimodal mapping of subcellular organization.Cell reports methods · 2026Article
- CRISPR Gene Tagging for Illuminating Endogenous Protein Dynamics.International journal of molecular sciences · 2026Review
- Strategies and considerations for the generation of ssDNA-Based HDR templates for CRISPR-based genome editing.BMC genomics · 2026Review
- Expression of Eukaryotic Membrane Proteins in Insect Cells.Methods in molecular biology (Clifton, N.J.) · 2026Article
- SIRT1 deacetylates GAPDH to drive microglial glycolysis and neuroinflammation.Frontiers in immunology · 2026Article
- Recent advances in nuclear actin research.Nucleus (Austin, Tex.) · 2025Review
- Homology-mediated transformation of frog-killing fungusProceedings of the National Academy of Sciences of the United States of America · 2025Article
- Insights into a water-mediated catalytic triad architecture in CE20 carbohydrate esterases.Nature communications · 2025Article
- Plant defense-directed discovery of a natural anti-psoriasis agent targeting GAPDH.Science advances · 2025Article
- Article
- Structural insights into polyisoprenyl-binding glycosyltransferases.Structure (London, England : 1993) · 2025Review
- Unraveling atomic complexity from frozen samples.Structural dynamics (Melville, N.Y.) · 2025Article
- MYO1F regulates T-cell activation and glycolytic metabolism by promoting the acetylation of GAPDH.Cellular & molecular immunology · 2025Article
- Building up complexity in structural biology studies.Nature structural & molecular biology · 2024Article
- Mass Spectrometry-Based Proteomics Technologies to Define Endogenous Protein-Protein Interactions and Their Applications to Cancer and Viral Infectious Diseases.Mass spectrometry reviewsReview
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
Abstract
CRISPR/Cas9-based genome engineering has revolutionized our ability to manipulate biological systems, particularly in higher organisms. Here, we designed a set of homology-directed repair donor templates that enable efficient tagging of endogenous proteins with affinity tags by transient transfection and selection of genome-edited cells in various human cell lines. Combined with technological advancements in single-particle cryogenic electron microscopy, this strategy allows efficient structural studies of endogenous proteins captured in their native cellular environment and during different cellular processes. We demonstrated this strategy by tagging six different human proteins in both HEK293T and Jurkat cells. Moreover, analysis of endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HEK293T cells allowed us to follow its behavior spatially and temporally in response to prolonged oxidative stress, correlating the increased number of oxidation-induced inactive catalytic sites in GAPDH with its translocation from cytosol to nucleus.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.