Understanding the expression, function and dynamics of proteins in their native environment is a fundamental goal that’s common to diverse aspects of molecular and cell biology. To study a protein, it must first be labeled—either directly or indirectly—with a “tag” that allows specific and sensitive detection.
Using a labeled antibody to the protein of interest is a common method to study native proteins. However, antibody-based assays, such as ELISAs and Western blots, are not suitable for use in live cells. These techniques are also limited by throughput and sensitivity. Further, suitable antibodies may not be available for the target protein of interest.
Transfecting a construct encoding a tagged protein into cultured cells or tissues is also a commonly used approach that simplifies protein detection. Depending on the tag used, it can even enable live-cell analysis. However, transfection methods remove the protein from its native genomic context and can result in nonphysiological levels of the expressed protein. Consequently, the protein stoichiometry with its interacting partners is altered, complicating the study of protein-protein interactions or kinetic analysis of protein abundance. In some cases, the tag used may also interfere with target protein function.
The refinement of CRISPR/Cas9-based gene editing technology over the past few years has offered a viable alternative to antibody-based and overexpression methods for studying endogenous proteins. (For an overview of CRISPR/Cas9 gene editing, see this blog post.) Using CRISPR/Cas9 systems, it’s possible to “knock in” a tag at a precise location in the genome, creating either an amino (N)-terminal or carboxy (C)-terminal fusion with the target protein. This method enables the study of endogenous proteins expressed under the regulation of native promoters and enhancers, providing a true picture of their behavior within a cell. Recently, Promega signed a license agreement with MilliporeSigma to access foundational CRISPR integration technology, creating new opportunities for the study of endogenous biology, particularly in the context of drug discovery and development.
CRISPR-Based Tagging Options
Ideally, a protein tag should be small, not subject to post-translational modifications, and enable highly sensitive detection with a large dynamic range. The HiBiT tag is a small, 11 amino acid peptide that binds with high affinity to a larger subunit called LgBiT that can be added to the assay medium or expressed in cells. The bound complex has high luciferase enzyme activity and will generate a bright luminescent signal in the presence of substrate, allowing relative protein quantitation over 7 logs of linear dynamic range. Alternatively, instead of HiBiT:LgBiT complementation, NanoLuc® Luciferase can be used as a tag that generates a comparably bright signal and, at 19KDa, is smaller than firefly luciferase.
There are two approaches to using CRISPR/Cas9 gene editing to knock in a bioluminescent tag:
- Create your own knock-in cell line.
- Take advantage of Promega-engineered cell lines and clones, available for a variety of target proteins and cell backgrounds.
Applications of CRISPR Knock-in Tagging
Introducing a bioluminescent tag with CRISPR/Cas9 simplifies the study of endogenous proteins in several areas of drug discovery research and development.
Targeted Protein Degradation: Selectively targeting proteins for removal from the cell, instead of inhibiting protein activity, is a newer modality for potential therapy. HiBiT technology provides quantitative measurements of protein degradation by enabling real-time analysis of degrader compounds in a high-throughput format.
Target Engagement: NanoLuc® fusion proteins and fluorescent tracers offer highly sensitive bioluminescence resonance energy transfer (BRET) detection, which simplifies measuring compound-target residence time and IC50 values.
Protein Internalization and Secretion: The Nano-Glo® HiBiT Extracellular Detection System quantitates HiBiT-tagged cell-surface or secreted protein expression in live cells within minutes, using a simple add-mix-read assay format.
Learn more about using CRISPR/Cas9 to knock in bioluminescent tags with this free webinar.
Latest posts by Ken Doyle (see all)
- RAS-Targeted Drug Discovery: From Challenge to Opportunity - June 28, 2021
- Endpoint PCR in 15 Minutes with New Master Mix and Thermal Cycler Combination - June 8, 2021
- COVID-19 Therapies: Are We There Yet? - March 24, 2021