CRISPR/Cas9-Enabled Endogenous Biology
By using CRISPR/Cas9 gene editing to knock in a bioluminescent tag at endogenous loci of interest, proteins expressed by the native promoter can be studied using simple light output measurements. Compared to overexpression models, endogenously tagged proteins maintain natural epigenetic regulation and stoichiometry with native interacting partners, resulting in improved assay windows, minimized artifacts, and more accurate models of protein biology under physiologically relevant conditions.
HiBiT is a popular tag for studying endogenous proteins. HiBiT is a small, 11 amino acid peptide that binds with high affinity to a larger subunit called LgBiT. The bound complex has high luciferase activity and will generate a bright luminescent signal in the presence of added substrate.
Introduction: Tagging Endogenous Loci Using CRISPR/Cas9 Knock-in
Using the combination of CRISPR/Cas9 knock-in and live-cell bioluminescent detection methods, changes in protein abundance can be monitored in real time to better understand cellular protein dynamics.
Why use endogenous tagging?
- Monitor physiological expression levels: no gene dosage effect
- Study genes under control of a native promoter and epigenetic regulation
- Maintain stoichiometry with native interacting partners
Determining Regulated Protein Abundance
Cellular protein levels are naturally modulated by changing cellular conditions, or they can be purposefully manipulated as a therapeutic strategy. Recently, targeting proteins for degradation has emerged as a new small molecule modality, enabling previously undruggable targets to be addressed. Using CRISPR/Cas9 gene editing to endogenously insert bioluminescent tags enables quantitative assays for protein abundance. These assays are sensitive and easy to read, in end-point or live-cell formats.
Promega offers a comprehensive selection of CRISPR-edited cell line pools and clones for popular targets and cell backgrounds:
Endogenous tagging creates an improved assay window for HIF1A abundance assay. HeLa cells were transiently transfected with different amounts of CMV- or PGK-driven expression constructs for Hif1a-HiBiT; alternatively, HiBiT was tagged to the endogenous locus in HeLa cells using CRISPR/Cas9 gene editing. Cells were treated with a titration of 1,10-phenanthroline as indicated and protein abundance measured using the Nano-Glo® HiBiT Lytic Reagent.
Degradation kinetics of endogenous HiBiT-BRD4 following PROTAC treatment. HiBiT was inserted at the endogenous BRD4 locus in HEK293 cells stably expressing LgBiT. Cells were treated with a titration of MZ1 in CO2-independent medium containing Nano-Glo® Endurazine™ substrate.
Internalization of Cell Surface Receptors
Following ligand binding, cell surface receptors often undergo internalization as a mechanism for signal regulation. Using CRISPR/Cas9 gene editing to insert the bioluminescent tag at the extracellular terminus of the receptor, internalization and subsequent trafficking back to the cell surface can be monitored.
Quantification of endogenous membrane receptor internalization following compound treatment. Pools of HeLa cells that were previously CRISPR-edited to insert HiBiT at the endogenous EGFR locus were treated with EGF as indicated. The HiBiT Extracellular Detection System was used to specifically detect HiBiT-EGFR expressed at the cell surface.
Downstream Target Gene Regulation
Changes in the abundance or activity of proteins upstream in a signaling pathway often result in modulation of downstream target gene expression. Using CRISPR/Cas9 gene editing, endogenous downstream target genes can be tagged and the expression of their corresponding proteins examined to understand the impacts on target gene regulation. Using live-cell assays, an abundance of downstream targets can be monitored in real time.
CRISPR Knock-in Process
The CRISPR knock-in process is a simple and efficient method for HiBiT tagging that requires no molecular cloning steps. From gene editing to assaying endogenous biology, the process can be completed in as little as 24 hours.
Choosing a workflow for CRISPR-enabled endogenous biology experiments will depend on the type of experiment, laboratory resources and experience, and the project timeline. You can design your experiment yourself from start to finish, or you can work with Promega to take advantage of our CRISPR-edited cell line pools and clones, available for a variety of target proteins and cell backgrounds. If you need a target or cell line not currently available, our Custom Development team will be happy to assist you.
Option 1: Do It Yourself (DIY)
We can walk you through the process of creating CRISPR knock-ins, step by step.
Option 2: Work with Promega
More Applications of CRISPR Knock-in Gene Editing
The versatility and convenience of the CRISPR knock-in process enable its use in a wide range of applications. Key examples are illustrated here.
Targeted Protein Degradation
Selectively targeting proteins for removal from the cell is a promising new modality for potential therapy.
Discover sensitive assays to measure compound binding at select target proteins in intact cells.
CRISPR/Cas9-Enabled Endogenous Biology Resources
Studying Endogenous Protein Dynamics with CRISPR-Mediated Tagging: Understanding Your Options
A comprehensive discussion of methods for knock-in of protein tags, and results of assays to measure endogenous protein function.
CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide
This ACS Chem. Biol. publication demonstrates the ability to efficiently tag endogenous proteins with a small luminescent peptide. The method allows sensitive quantitation of protein response dynamics in regulated expression and with covalent modifications.
HiBiT Detection Reagents
A comparison of several methods to detect and quantify any tagged protein with a simple, luminescent signal.