Duplex Labeling and Manipulation of Neuronal Proteins Using Sequential CRISPR/Cas9 Gene Editing

Area of Science:

• Neuroscience
• Molecular Biology
• Genetics

Background:

• CRISPR/Cas9 knock-in methods allow endogenous protein labeling for studying spatiotemporal distribution.
• Multiplexing knock-in events in neurons is challenging due to crosstalk between editing events.

Purpose of the Study:

• To develop an efficient, flexible, and accurate method for multiplex genome editing in rat neurons.
• To overcome crosstalk limitations in CRISPR/Cas9 multiplexing for neuronal protein labeling.

Main Methods:

• Conditional Activation of Knock-in Expression (CAKE) utilizes sequential, recombinase-driven guide RNA expression.
• CAKE controls the timing of genomic integration for each donor sequence to minimize crosstalk.
• Super-resolution microscopy and inducible dimerization modules were employed.

Main Results:

• CAKE enables efficient and accurate multiplex genome editing in rat neurons.
• The method successfully co-labeled various endogenous neuronal proteins, including cytoskeletal proteins, synaptic scaffolds, ion channels, and receptor subunits.
• Synaptic protein co-organization was found to depend on synapse size, and receptor dynamics were acutely controlled.

Conclusions:

• CAKE is a versatile method for multiplex protein labeling, detection, localization, and manipulation of endogenous proteins in neurons.
• The developed strategy provides new biological insights into neuronal function and protein organization.
• CAKE facilitates accurate duplex endogenous protein labeling and manipulation for addressing complex biological questions.