CRISPR deletions in cell lines for reconstitution studies of pseudokinase function
- Author(s)
- Jacobsen, Annette V; Murphy, James M;
- Journal Title
- Methods in Enzymology
- Abstract
- The non-catalytic cousins of protein kinases, the pseudokinases, have grown to prominence as indispensable signaling entities over the past decade, despite their lack of catalytic activity. Because their importance has only been fully embraced recently, many of the 10% of the human kinome categorized as pseudokinases are yet to be attributed biological functions. The advent of CRISPR-Cas9 editing to genetically delete pseudokinases in a cell line of interest has proven invaluable to dissecting many functions and remains the method of choice for gene knockout. Here, using the terminal effector pseudokinase in the necroptosis cell death pathway, MLKL, as an exemplar, we describe a method for genetic knockout of pseudokinases in cultured cells. This method does not retain the CRISPR guide sequence in the edited cells, which eliminates possible interference in subsequent reconstitution studies where mutant forms of the pseudokinase can be reintroduced into cells exogenously for detailed mechanistic characterization.
- Publisher
- Academic Press
- Keywords
- MLKL; Pseudokinase; CRISPR; Gene editing; Complementation
- Research Division(s)
- Inflammation
- PubMed ID
- 35525543
- Publisher's Version
- https://doi.org/10.1016/bs.mie.2022.03.054
- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2022-05-11 10:50:29
Last Modified: 2022-05-11 11:22:03