Massengill, C. I. | Search bioRxiv

bioRxiv2021

Highly sensitive genetically-encoded sensors for population and subcellular imaging of cAMP in vivo

Cyclic adenosine monophosphate (cAMP) integrates information from diverse G protein-coupled receptors, such as neuromodulator receptors, to regulate pivotal biological processes in a cellular- and subcellular-specific manner. However, in vivo cellular-resolution imaging of cAMP dynamics in neurons has not been demonstrated. Here, we screen existing genetically-encoded cAMP sensors, and further develop the best performer to derive three improved variants, called cAMPFIREs. These sensors exhibit up to ten-fold increased sensitivity to cAMP and a corrected, cytosolic distribution. cAMPFIREs are compatible with both ratiometric and fluorescence lifetime imaging, and can detect cAMP dynamics elicited by norepinephrine at physiologically-relevant, nanomolar concentrations. Imaging of cAMPFIREs in awake mice reveals tonic levels of cAMP in cortical neurons that are associated with wakefulness, and are differentially regulated in different subcellular compartments. Furthermore, enforced locomotion elicits neuron-specific, bidirectional cAMP dynamics, in part, mediated by norepinephrine. Finally, cAMPFIREs also function in Drosophila, suggesting that they have broad applicability for studying intracellular signaling in vivo.

neuroscience↗

bioRxiv2020

High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion

Precise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.

cell biology↗