A CRISPR-based Strategy for Temporally Controlled Site-Specific Editing of RNA Modifications

Chemical modifications on RNA play important roles in regulating its fate and various biological activities. However, the impact of RNA modifications varies depending on their locations on different transcripts and cells/tissues contexts; available tools to dissect context-specific RNA modifications are still limited. Herein, we report the detailed protocol for using a chemically inducible and reversible platform to achieve site-specific editing of the chosen RNA modification in a temporally controlled manner by integrating the clustered regularly interspaced short palindromic repeats (CRISPR) technology and the abscisic acid (ABA)-based chemically induced proximity (CIP) system. The procedures were demonstrated using the example of inducible and reversible N⁶-methyladenosine (m⁶A) editing and the evaluation of its impact on RNA properties with ABA addition and reversal with the control of ABA or light.