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CRISPR-Cas9 screen in NIH-3T3 cells to identify modulators of LRRK2 function
Output Details
Description
We describe here a pooled, CRISPR Cas9 screen to identify modulators of LRRK2 activity. LRRK2 phosphorylates a subset of Rab GTPases, thus LRRK2 activity can be monitored easily using phosphoRab-specific antibodies. Our CRISPR-Cas9 based screen is carried out in mouse cells using a ready-to-use pooled guide RNA (gRNA) mouse library consisting of 78,637 gRNAs targeting 19,674 genes and an extra 1,000 control gRNAs. This library is known as the Mouse Brie CRISPR knockout pooled library, and is a kind gift from David Root and John Doench (Addgene #73633). The protocols below are modifications of the approach described by Feng Zhang and colleagues (https://doi.org/10.1038/nprot.2017.016).
Identifier (DOI)
10.17504/protocols.io.8epv5jr9jl1b/v1