Yang, Xiaolu, Woldemichael, Kehali, Hover, John, Zhao, Shengli, Guo, Xiao, Qian, Yongjun, Gillis, Jesse, Huang, Z Josh (November 2024) RNA programmable cell targeting and manipulation with CellREADR. bioRxiv. ISSN 2692-8205
Abstract
Methods that provide specific, easy, and scalable experimental access to animal cell types and cell states 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' 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 highlights the potential for RNA-based monitoring and manipulation of animal cells in ways that are simple, versatile, and generalizable across tissues and species. Here, we describe a detailed protocol for implementing CellREADR experiments in cell cultures and in animals. The procedure includes sensor and payload design, cloning, validation and characterization in mammalian cell cultures. The in vivo animal protocol focuses on AAV-based delivery of CellREADR using brain tissue as examples. We describe current best practices, various experimental controls and trouble-shooting steps. Beginning from sensor design, the validation of RNA sensor-actuators in cell cultures can be completed in 2-3 weeks. The construction of AAV vectors and their in vivo validation and characterization will take additional 6-8 weeks.
| Item Type: | Paper |
|---|---|
| Subjects: | bioinformatics bioinformatics > genomics and proteomics bioinformatics > genomics and proteomics > Mapping and Rendering |
| CSHL Authors: | |
| Communities: | CSHL labs > Gillis Lab CSHL labs > Zador lab |
| SWORD Depositor: | CSHL Elements |
| Depositing User: | CSHL Elements |
| Date: | 26 November 2024 |
| Date Deposited: | 23 Dec 2024 16:27 |
| Last Modified: | 23 Dec 2024 16:27 |
| PMCID: | PMC11623600 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/41765 |
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