Multimodal CRISPR screens uncover DDX39B as a global repressor of A-to-I RNA editing
Tianzi Wei,
Jiaxuan Li,
Xiang Lei,
Risheng Lin,
Qingyan Wu,
Zhenfeng Zhang,
Shimin Shuai,
Ruilin Tian
Affiliations
Tianzi Wei
Department of Medical Neuroscience, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China; SUSTech Homeostatic Medicine Institute, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China; Key University Laboratory of Metabolism and Health of Guangdong, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China
Jiaxuan Li
SUSTech Homeostatic Medicine Institute, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China; Department of Human Cell Biology and Genetics, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China
Xiang Lei
Department of Medical Neuroscience, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China; SUSTech Homeostatic Medicine Institute, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China; Key University Laboratory of Metabolism and Health of Guangdong, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China
Risheng Lin
Department of Medical Neuroscience, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China; SUSTech Homeostatic Medicine Institute, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China; Key University Laboratory of Metabolism and Health of Guangdong, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China
Qingyan Wu
School of Public Health and Emergency Management, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China
Zhenfeng Zhang
School of Public Health and Emergency Management, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China
Shimin Shuai
SUSTech Homeostatic Medicine Institute, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China; Department of Human Cell Biology and Genetics, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China; Corresponding author
Ruilin Tian
Department of Medical Neuroscience, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China; SUSTech Homeostatic Medicine Institute, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China; Key University Laboratory of Metabolism and Health of Guangdong, Southern University of Science and Technology, Shenzhen, Guangdong 518055, China; Corresponding author
Summary: Adenosine-to-inosine (A-to-I) RNA editing is a critical post-transcriptional modification that diversifies the transcriptome and influences various cellular processes, yet its regulatory mechanisms remain largely unknown. Here, we present two complementary CRISPR-based genetic screening platforms: CREDITS (CRISPR-based RNA editing regulator screening), which enables genome-scale identification of editing regulators using an RNA recorder-based reporter system, and scCREDIT-seq (single-cell CRISPR-based RNA editing sequencing), which provides multiplexed single-cell characterization of transcriptome and editome changes for pooled perturbations. By screening 1,350 RNA-binding proteins, we identified a series of A-to-I editing regulators. Mechanistic investigation revealed DDX39B as a global repressor of A-to-I editing, which functions by preventing double-stranded RNA accumulation through its helicase activity. Targeting DDX39B significantly enhances the efficiency of RNA-editing-based tools, such as CellREADR (cell access through RNA sensing by endogenous ADAR) and LEAPER (leveraging endogenous ADAR for programmable editing of RNA), and disrupts hepatitis D virus (HDV) RNA editing homeostasis. These technological advances not only expand our understanding of RNA editing regulation but also provide powerful tools for exploring tissue-specific and context-dependent RNA modification mechanisms, with broad implications for therapeutic development.
