CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide
Open accessAbstract
Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (KD = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to quantify changes in abundance of the hypoxia inducible factor 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1α levels with the hydroxyproline modification that mediates its degradation. These results demonstrate the ability to efficiently tag endogenous proteins with a small luminescent peptide, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications.
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Other assay methodologies to detect target levels have been useful for the TPD field for high-throughput and accurate measurements, e.g. ELISA or the HiBit-LgBit split nanoluciferase system.
“Other assay methodologies to detect target levels have been very useful for the TPD field when aiming for highthroughput and accurate measurements (e.g., ELISA or the HiBit-LgBit split nanoluciferase system).164”
From Chasing molecular glue degraders: screening approaches (2022) · cites this paper as [164]
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- Authors
- Schwinn, M. K., Machleidt, T., Zimmerman, K., Eggers, C. T., Dixon, A. S., Hurst, R., Hall, M. P., Encell, L. P., Binkowski, B. F., & Wood, K. V.
- Affiliations
- Promega (United States) (US)
- University of Utah (US)
- Corresponding author
- Schwinn, Marie K.
- Venue
- ACS Chemical Biology
- Publisher
- American Chemical Society (ACS)
- Published
- 2017-09-11
- Volume
- 13
- Issue
- 2
- Pages
- 467-474
- ISSN
- 1554-8929 (ISSN-L)
- 1554-8937
- OpenAlex
- W2754886024
- Cited by
- 434
- References
- 30
- Citations by year
- 2026: 59 · 2025: 77 · 2024: 74 · 2023: 59 · 2022: 42 · 2021: 36 · 2020: 49 · 2019: 23 · 2018: 14 · 2017: 1
- FWCI
- 9.83
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- ACS Chemical Biology (publishedVersion, other-oa)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › bioluminescence and chemiluminescence research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Keywords
- HiBiT, CRISPR/Cas9 genome editing, endogenous protein tagging, HIF-1α, hypoxia, protein degradation, transcriptional regulation, intracellular signaling
- MeSH terms
- Gene Editing, CRISPR-Associated Protein 9, Antibodies, Carrier Proteins, HeLa Cells, Humans, Leupeptins, Luciferases, Luminescent Proteins, Membrane Proteins, Neoplasm Proteins, Oligopeptides, Protein Processing, Post-Translational, Proto-Oncogene Proteins, Streptococcus pyogenes, Genes, Reporter, Low Density Lipoprotein Receptor-Related Protein-2, Adaptor Proteins, Signal Transducing, Luminescence, Kruppel-Like Transcription Factors, Early Growth Response Transcription Factors, Hypoxia-Inducible Factor 1, alpha Subunit, Bioluminescence Resonance Energy Transfer Techniques, CRISPR-Cas Systems
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