Protocol for the generation of DDT signaling reporter cell line for CRISPR screening

  • STAR Protoc. 2026 Jun 19;7(2):104523. doi: 10.1016/j.xpro.2026.104523.
Zhengjin He  1 ,  Shishuang Chen  1 ,  Aowei Huang  1 ,  Hai Jiang  2
Affiliations
  • 1. Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China.
  • 2. Key Laboratory of RNA Innovation, Science and Engineering, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China; Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China. Electronic address: [email protected].
PMID: 42033729 DOI: 10.1016/j.xpro.2026.104523
Abstract

Cells respond to perturbations through signaling pathways that often induce characteristic transcriptional changes. Here, we present a protocol for generating a double death trap (DDT) reporter that converts pathway activity into a binary survival-death outcome. The DDT reporter employs puromycin resistance and FKBP12(F36V)-ΔCaspase9 constructs driven by pathway-specific response elements. We describe the steps for DDT reporter plasmid construction, cell line generation, and genome-wide CRISPR screening in DDT cells. We further detail procedures for next-generation Sequencing (NGS) sample preparation, Sequencing, and downstream analysis. For complete details on the use and execution of this protocol, please refer to He et al.1.

Keywords
Biotechnology and bioengineering; CRISPR; Cell Biology.
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