The ability to precisely control gene expression using small-molecule drugs is a valuable tool in research and has important therapeutic potential. However, existing systems are often limited by the toxicity of the drugs and the need to alter gene sequences or endogenous regulatory elements. Here, we introduce Cyclone (acyclovir-controlled poison exon), an acyclovir-controlled poison exon cassette that can be used for small-molecule control of both transgene and endogenous gene expression. Cyclone is a portable ‘intron–poison exon–intron’ element that can be inserted into nearly any gene and is completely removed upon acyclovir treatment, leaving the native transcript intact. Cyclone offers tunable, reversible gene expression with nearly undetectable background and a ~295-fold activation. We also present Pac-Cyclone, a cassette that simplifies the generation of cell lines with acyclovir-controlled endogenous gene expression. Finally, we demonstrate the programmability of Cyclone, underscoring its potential for developing diverse genetic circuits controlled by various ligands.
