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Title: Production of CRISPR‐Cas9 Transgenic Cell Lines for Knocksideways Studies
Abstract

Protein activity is generally functionally integrated and spatially restricted to key locations within the cell. Knocksideways experiments allow researchers to rapidly move proteins to alternate or ectopic regions of the cell and assess the resultant cellular response. Briefly, individual proteins to be tested using this approach must be modified with moieties that dimerize under treatment with rapamycin to promote the experimental spatial relocalizations. CRISPR technology enables researchers to engineer modified protein directly in cells while preserving proper protein levels because the engineered protein will be expressed from endogenous promoters. Here we provide straightforward instructions to engineer tagged, rapamycin‐relocalizable proteins in cells. The protocol is described in the context of our work with the microtubule depolymerizer MCAK/Kif2C, but it is easily adaptable to other genes and alternate tags such as degrons, optogenetic constructs, and other experimentally useful modifications. Off‐target effects are minimized by testing for the most efficient target site using a split‐GFP construct. This protocol involves no proprietary kits, only plasmids available from repositories (such as addgene.org). Validation, relocalization, and some example novel discoveries obtained working with endogenous protein levels are described. A graduate student with access to a fluorescence microscope should be able to prepare engineered cells with spatially controllable endogenous protein using this protocol. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Choosing a target site for gene modification

Basic Protocol 2: Design of gRNA(s) for targeted gene modification

Basic Protocol 3: Split‐GFP test for target efficiency

Basic Protocol 4: Design of the recombination template and analytical primers

Support Protocol 1: Design of primers for analytical PCR

Basic Protocol 5: Transfection, isolation, and validation of engineered cells

Support Protocol 2: Stable transfection of engineered cells with binding partners

 
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NSF-PAR ID:
10483819
Author(s) / Creator(s):
 ;  ;  ;  
Publisher / Repository:
Wiley Blackwell (John Wiley & Sons)
Date Published:
Journal Name:
Current Protocols
Volume:
3
Issue:
12
ISSN:
2691-1299
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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    Support Protocol 5: Estimating biotinylation percentage by streptavidin precipitation

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