Scalable recombinase-based gene expression cascades

Abstract

Temporal modulation of multiple genes underlies sophisticated biological phenomena. However, there are few scalable and generalizable gene circuit architectures for the programming of sequential genetic perturbations. 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 used tandem GPCs to sequentially express single-guide RNAs to encode transcriptional cascades and trigger the sequential accumulation of mutations. We built an all-inone gene circuit that sequentially edits genomic loci, synchronizes cells at a specific stage within