PRJNA560978
Massively Parallel RNA Device Engineering in Mammalian Cells with RNA-seq
Synthetic RNA-based gene control devices dynamically control a wide range of gene-regulatory processes across diverse cell types. However, the limited throughput of quantitative assays in mammalian cells has hindered fast iteration and interrogation of sequence space needed to identify new RNA devices. To address this challenge, we developed a quantitative, rapid and high-throughput mammalian cell-based RNA-Seq assay to efficiently engineer RNA devices. We identified new ribozyme-based RNA devices that respond to theophylline, hypoxanthine, cyclic-di-GMP, and folinic acid from libraries of ~22,700 sequences in total. The small molecule-responsive devices exhibit low basal expression and high activation ratios, significantly expanding our toolset of highly functional ribozyme switches. The large datasets obtained further provided conserved sequence and structure motifs that may be used for rationally  guided design. The RNA-Seq approach offers a generally applicable strategy for developing broad classes of RNA devices, thereby advancing the engineering of genetic devices for mammalian systems.