Frequently Asked Questions
HiBiT-Based Endogenous Tagging with CRISPR Knock-In Cell Lines
A detailed workflow for design, editing, and validation of CRISPR knock-in cell lines can be found here: CRISPR-Based Endogenous Tagging Application Note.
How can CRISPR-based tagging support my research?
Endogenous tagging provides a versatile platform for studying proteins in their native context. By incorporating small, functional tags directly into the genome, you can explore a wide range of biological questions, including:
- Quantitative studies of protein expression and turnover.
- Real-time monitoring of protein localization and trafficking.
- Protein–protein interaction assays.
- Biologic drug discovery and validation studies.
- Comparative studies of tagged proteins in engineered vs. patient-derived cell models (e.g., iPSCs, disease lines).
New to CRISPR knock-ins? Start with our overview page for the basics.
How do I choose the right tagging strategy (N- vs. C-terminal, internal)?
- N-terminal: Place immediately after the start codon, or after a signal peptide if present. A VS linker may be beneficial to include between the signal peptide and the HiBiT tag.
- C-terminal: Insert just before the stop codon.
- Internal: Target flexible loops or unstructured regions to minimize disruption of function. If functionality is unknown, test both termini or include flexible linkers to reduce interference.
How does CRISPR-based integration differ from vector integration?
Both approaches can be used to introduce new genetic material into cells, but they differ in how the DNA is integrated and maintained within the genome.
Vector-based integration:
- Relies on viral or nonviral vectors to deliver DNA into the genome.
- Integration occurs through random insertion events, making the insertion site unpredictable.
- Expression levels can vary or diminish over time as cells divide or silence the inserted sequence.
CRISPR-based integration:
- Enables precise, site-specific addition of genes under native regulatory control.
- Uses homology-directed repair to make the new gene a stable, permanent part of the genome.
- Maintains consistent expression and removes the need to remake tagged cell lines for each experiment.
Does the tag affect protein function?
HiBiT and other small protein tags are designed to minimize disruption, but placement has the potential to impact folding, trafficking or interactions. To ensure minimal interference, it’s important to I) design thoughtfully and II) validate the results.
- Design to minimize interference: Place the tag at the N- or C-terminus, or within flexible regions that are less likely to disrupt folding or activity and use short linkers to preserve protein structure and accessibility.
- Validate that native function is maintained: Compare tagged and untagged versions to confirm that biological activity remains consistent; monitor localization and trafficking in real time to verify proper cellular behavior.
What can I do to improve knock-in efficiency?
- Use homology-directed repair (HDR) donors with optimized homology arms.
- Incorporate silent PAM mutations in the donor to prevent Cas9 recutting.
- Consider using DNA repair inhibitors (e.g., DNA-PK and Pol Theta inhibitors) to suppress competing repair pathways and boost HDR integration.
- Delivery method matters: electroporation typically gives higher efficiency than lipid-based transfection.
How do I confirm that my tag was integrated and is functioning correctly?
Validation typically involves combining functional assays, molecular confirmation and protein-level characterization. It’s suggested to periodically validate the stability of your tagged lines. Here are specific approaches for validation:
- Functional detection:
- Molecular validation:
- PCR or droplet digital PCR to determine presence and zygosity (homozygous vs heterozygous)
- Sanger sequencing to confirm sequence fidelity
- Protein validation: Antibody-based methods (Western blot, IP, IF, FACS) can confirm expression and localization
Can I multiplex by tagging multiple proteins in one cell line?
Yes, but with caveats:
- Different proteins can be tagged with distinct reporters (e.g., HiBiT and HaloTag) to allow orthogonal readouts.
- Simultaneous knock-ins are possible, but efficiency decreases with each additional target.
- For protein–protein interactions, the NanoBiT® PPI Starter System allows split tagging for complementation assays.
For more information about choosing a tag for your protein, check out this article.
What types of cells work best for CRISPR-based tagging?
Many standard immortalized cell lines are well suited for CRISPR-based endogenous tagging and generally provide reliable results. However, knock-in efficiency can vary significantly depending on the cell type, the genomic locus and the size of the tag being introduced.
More challenging models like primary cells, induced pluripotent stem cells (iPSCs) or neurons often require additional optimization of delivery methods and careful strategies for selecting edited populations. In cases where expression of the target protein is low, or when long-term stability of the edit is needed, clonal isolation is strongly recommended. This ensures that the integrated tag is stably inherited and that downstream experiments are not confounded by mixed populations of edited and unedited cells.
Looking for ready-to-use cell lines? Check out our catalog of ready-to-use cell line pools and clones.
For additional information, view our HiBiT-Powered CRISPR Knock-Ins for Endogenous Tagging resource page.
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