Scalable recombinase-based gene expression cascades.

Temporal modulation of the expression of multiple genes underlies complex complex biological phenomena. However, there are few scalable and generalizable gene circuit architectures for the programming of sequential genetic perturbations. Here, we describe a modular recombinase-based gene circuit architecture, comprising tandem gene perturbation cassettes (GPCs), that enables the sequential expression of multiple genes in a defined temporal order by alternating treatment with just two orthogonal ligands. We use tandem GPCs to sequentially express single-guide RNAs to encode transcriptional cascades that trigger the sequential accumulation of mutations. We build an all-in-one gene circuit that sequentially edits genomic loci, synchronizes cells at a specific stage within a gene expression cascade, and deletes itself for safety. Tandem GPCs offer a multi-tiered cellular programming tool for modeling multi-stage genetic changes, such as tumorigenesis and cellular differentiation.