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bioRxiv · 10.1101/2022.01.02.474730

Multiplex labeling and manipulation of endogenous neuronal proteins using sequential CRISPR/Cas9 gene editing

Droogers, W. J.·Willems, J.·MacGillavry, H. D.·de Jong, A. P.
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Abstract

Recent advances in CRISPR/Cas9-mediated knock-in methods enable labeling of individual endogenous proteins with fluorophores, to determine their spatiotemporal expression in intact biological preparations. However, multiplex knock-in methods remain limited, particularly in postmitotic cells, due to a high degree of crosstalk between genome editing events. We present Conditional Activation of Knock-in Expression (CAKE), which delivers efficient, flexible and accurate multiplex genome editing in neurons. CAKE is based on sequential gRNA expression operated by a Cre- or Flp-recombinase to control the time window for genomic integration of each donor sequence, which diminishes crosstalk between genome editing events. Importantly, CAKE is compatible with multiple CRISPR/Cas9 strategies, and we show the utilization of CAKE for co-localization of various endogenous proteins, including synaptic scaffolds, ion channels and neurotransmitter receptor subunits. Knock-in efficacy was highly sensitive to DNA vector amount, while knock-in crosstalk was dependent on the rate of donor DNA integration and timing of Cre activation. We applied CAKE to study the co-distribution of endogenous synaptic proteins using dual-color single-molecule localization microscopy, and we introduced dimerization modules to acutely control synaptic receptor dynamics in living neurons. Taken together, CAKE is a versatile method for multiplex protein labeling, enabling accurate detection, precise localization and acute manipulation of endogenous proteins in single cells.

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Droogers, W. J., Willems, J., MacGillavry, H. D., de Jong, A. P.. 2022-01-02. Multiplex labeling and manipulation of endogenous neuronal proteins using sequential CRISPR/Cas9 gene editing. https://doi.org/10.1101/2022.01.02.474730

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