CellREADR: An ADAR-based RNA sensor-actuator device

RNAs are central mediators of genetic information flow and gene regulation that underlie diverse cell types and cell states across species. Thus, methods that can sense and respond to RNA profiles in living cells will have broad applications in biology and medicine. CellREADR - Cell access through RNA sensing by Endogenous ADAR (adenosine deaminase acting on RNA), is a programmable RNA sensor-actuator technology that couples the detection of a cell-defining RNA to the translation of an effector protein to monitor and manipulate the cell. The CellREADR RNA device consists of a 5' sensor region complementary to a cellular RNA and a 3' protein payload coding region. Payload translation is gated by the removal of a STOP codon in the sensor region upon base pairing with the cognate cellular RNA through an ADAR-mediated A-to-I editing mechanism ubiquitous to metazoan cells. CellREADR thus represents a new generation of programmable RNA device for monitoring and manipulating animal cells in ways that are simple, versatile, and generalizable across tissues and species. Here, we describe a detailed procedure for implementing CellREADR experiments in cell culture systems and in animals. The procedure includes sensor and payload design, cloning, validation and characterization in mammalian cell cultures. The in vivo protocol focuses on AAV-based delivery of CellREADR through expression vectors using brain tissue as an example. We describe current best practices and various experimental controls.