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CRISPR deletions in cell lines for reconstitution studies of pseudokinase function
Annette V Jacobsen1, James M Murphy1
1Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia.
Methods in Enzymology
|May 7, 2022
Summary
Researchers developed a new CRISPR-Cas9 method for gene knockout of pseudokinases, like MLKL, in cultured cells. This technique removes the guide sequence, aiding future studies on pseudokinase function and signaling pathways.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Pseudokinases are crucial signaling proteins lacking catalytic activity, with many yet to have their functions elucidated.
- The human kinome includes approximately 10% pseudokinases, highlighting their broad biological significance.
- CRISPR-Cas9 gene editing is a powerful tool for studying pseudokinase functions via gene knockout.
Purpose of the Study:
- To present an improved CRISPR-Cas9 method for generating pseudokinase knockouts in cultured cells.
- To utilize MLKL, a key pseudokinase in necroptosis, as a model for this gene knockout technique.
- To develop a method that avoids guide sequence retention for subsequent molecular studies.
Main Methods:
- Employing CRISPR-Cas9 gene editing to achieve genetic deletion of pseudokinases.
- Utilizing MLKL, the terminal effector pseudokinase in necroptosis, as an exemplar.
- Implementing a strategy to ensure the CRISPR guide sequence is not retained in edited cells.
Main Results:
- Successful genetic knockout of pseudokinases in cultured cells was achieved.
- The developed method ensures the absence of the CRISPR guide sequence post-editing.
- This facilitates future reconstitution studies for mechanistic characterization.
Conclusions:
- The described CRISPR-Cas9 method provides a robust approach for pseudokinase gene knockout.
- Eliminating guide sequence retention is critical for accurate downstream functional analysis.
- This technique advances the study of pseudokinase roles in cellular signaling and pathways like necroptosis.
