CRISPR DIY Overview
If you plan to design your own CRISPR-enabled workflow to study endogenous proteins using HiBiT tagging, you don’t have to go it alone. Take advantage of our in-depth experience to make sure you get the results you need.
Summary of the CRISPR DIY Process
Before creating your knock-in cell line, you will need to perform some genomic analysis to determine the ideal location for the Cas9 cut site and design the donor DNA that will recombine to insert the HiBiT tag into the desired genomic location. Here are the basic steps you will follow along with more detailed guidance on the process.
- Design and order your guide RNA, donor DNA and Cas9.
- Obtain rights to synthesize the HiBiT tag.
- Work with an oligo provider to obtain the required materials.
- Perform the gene-editing step to add the HiBiT tag to the target of interest.
- Assay the cell pool or move forward with clone isolation.
Promega scientists continue to study the use of CRISPR-Cas9 for endogenously tagging proteins with HiBiT. Read about our recent research project that shows how HiBiT knock-in tagging is a broadly applicable and scalable method to analyze endogenous proteins and demonstrates the advantages of endogenous tagging compared to overexpression models.
Endogenous HiBiT tagging of proteins with diverse structure and function. CRISPR/Cas9 gene editing was used to HiBiT tag the endogenous loci of genes representing various cellular functions, as indicated. Bioluminescent imaging was performed to verify that expressed HiBiT-tagged proteins accurately reflect natural biology by displaying the expected subcellular localization.
Study Endogenous Protein Dynamics
Expressing intracellular LgBiT protein for HiBiT analysis allows you to measure real-time abundance of endogenously regulated HiBiT-tagged proteins, as well as use complemented HiBiT as the energy donor in NanoBRET™ applications for even more assay possibilities.
- Perform live-cell, kinetic analysis of HiBiT-tagged proteins; no cell lysis required
- Compatible with exogenously or endogenously expressed HiBiT fusion proteins
- Use complemented HiBiT as the energy donor for live-cell NanoBRET™ applications
Several tools are available to incorporate intracellular LgBiT into your CRISPR/Cas9 HiBiT knock-in workflow.
Degradation kinetics of endogenous HiBiT-BRD4 following PROTAC treatment. HiBiT was inserted at the endogenous BRD4 locus in the HEK293 LgBiT Cell Line. Cells were treated with a titration of MZ1 in CO2-independent medium containing Nano-Glo® Endurazine™ substrate.
CRISPR DIY Resources
A Practical Guide to CRISPR-Mediated Gene Tagging with a Bioluminescent Peptide
Learn about a simple and efficient method for CRISPR-mediated HiBiT tagging that requires no molecular cloning steps, taking you from gene editing to assaying endogenous biology in as little as 24 hours
How to Tag Proteins with HiBiT
Discover how to detect and quantify any tagged protein with a simple, luminescent signal and learn about applications and detection format options.
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.