Generation of locus-specific degradable tag knock-ins in mouse and human cell lines

Damhofer H, Radzisheuskaya A, Helin K.
Source: STAR Protoc
Publication Date: (2021)
Issue: 2(2): 100575
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Basic Research
Molecular Biology
Cells used in publication:
Species: human
Tissue Origin: blood
Embryonic stem cell (ES), mouse
Species: mouse
Tissue Origin: embryo
Species: human
Tissue Origin: blood
Hematopoietic stem cells, mouse
Species: mouse
Tissue Origin: bone marrow
4D-Nucleofector® X-Unit

d. Nucleofect RNP complex and donor plasmid into recipient cells using Amaxa 4D-nucleofector (Lonza). The choice of nucleofection program and solution is cell line-dependent and requires optimization beforehand. For commonly used cell lines, optimized nucleofection conditions are found on the Lonza website. The nucleofection programs for cell types we have successfully used in the lab are provided in the materials and equipment section.

i. Mix 16.4 mL cell line specific nucleofection buffer (e.g., P3 Primary Cell) with 3.6 mL buffer supplement.

ii. Add 500 ng pUC19 donor plasmid (in the smallest volume possible) to 20 µL nucleofection buffer mix.

iii. Resuspend 2x10^5 cells in nucleofection buffer/plasmid mix.

iv. Transfer cell suspension to tube with assembled RNP complex and mix solutions by carefully pipetting up and down with a P200 pipette tip. v. Transfer nucleofection mix into 16-well Nucleocuvette strip.


Protein degradation technologies represent a powerful functional genomics tool, allowing fast and controllable target protein depletion. Establishing these systems requires a knock-in of the degradation tag into both endogenous target gene alleles. Here, we provide a step-by-step protocol for the efficient generation of biallelic degradation tag knock-ins in mouse and human cell lines using CRISPR-Cas9. We use knockin of an endogenous Kansl3 degradation tag in mouse embryonic stem (ES) cells as an example but provide modifications for application in other cell types. For complete details on the use and execution of this protocol, please refer to Radzisheuskaya et al. (2021).