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Duplex Labeling and Manipulation of Neuronal Proteins Using Sequential CRISPR/Cas9 Gene Editing.
| Content Provider | Europe PMC |
|---|---|
| Author | Droogers, Wouter J. Willems, Jelmer MacGillavry, Harold D. de Jong, Arthur P. H. |
| Copyright Year | 2022 |
| Abstract | CRISPR/Cas9-mediated knock-in methods enable the labeling of individual endogenous proteins to faithfully determine their spatiotemporal distribution in cells. However, reliable multiplexing of knock-in events in neurons remains challenging because of cross talk between editing events. To overcome this, we developed conditional activation of knock-in expression (CAKE), allowing efficient, flexible, and accurate multiplex genome editing. To diminish cross talk, CAKE is based on sequential, recombinase-driven guide RNA (gRNA) expression to control the timing of genomic integration of each donor sequence. We show that CAKE is broadly applicable in rat neurons to co-label various endogenous proteins, including cytoskeletal proteins, synaptic scaffolds, ion channels and neurotransmitter receptor subunits. To take full advantage of CAKE, we resolved the nanoscale co-distribution of endogenous synaptic proteins using super-resolution microscopy, demonstrating that their co-organization correlates with synapse size. Finally, we introduced inducible dimerization modules, providing acute control over synaptic receptor dynamics in living neurons. These experiments highlight the potential of CAKE to reveal new biological insight. Altogether, CAKE is a versatile method for multiplex protein labeling that enables the detection, localization, and manipulation of endogenous proteins in neurons. |
| Page Count | 21 |
| Journal | eNeuro |
| PubMed Central reference number | PMC9333357 |
| Issue Number | 4 |
| PubMed reference number | 35851300 |
| e-ISSN | 23732822 |
| DOI | 10.1523/ENEURO.0056-22.2022 |
| Language | English |
| Publisher | Society for Neuroscience |
| Publisher Date | 2022-07-18 |
| Access Restriction | Open |
| Rights License | This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. Copyright © 2022 Droogers et al. |
| Subject Keyword | CRISPR/Cas9 fluorescence microscopy knock-in multiplex genome editing neurons |
| Content Type | Text |
| Resource Type | Article |
| Subject | Medicine Neuroscience |
