This protocol establishes a double death trap (DDT) reporter that converts signaling pathway activity into a binary survival-death readout.
The DDT reporter combines a puromycin resistance cassette driven by pathway-specific response elements with an FKBP12(F36V)-ΔCaspase9 suicide switch.
The protocol covers the full workflow:
- Construction of the DDT reporter plasmid
- Generation of the reporter cell line
- Genome-wide CRISPR screening in DDT cells
- Next-generation sequencing (NGS) sample preparation, sequencing, and downstream analysis
For complete details on the use and execution of this protocol, please refer to the companion research paper, STT3A is essential for Wnt signaling… (He et al.).