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.
He et al. (Fri,) studied this question.