A portable poison exon for small molecule control of mammalian gene expression

The ability to precisely control gene expression using small molecule drugs is a valuable tool in research and has significant 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. HEK293T cells (ATCC CRL-3216) were maintained at 37 °C in a humidified 5 % CO₂ incubator in Dulbecco’s Modified Eagle Medium (DMEM, Thermo 11995065) supplemented with 10 % heat-inactivated fetal bovine serum, 100 U mL⁻¹ penicillin, and 100 µg mL⁻¹ streptomycin. Cells were sub-cultured at 70–80 % confluence by trypsinisation, counted with a TC20™ Automated Cell Counter, and seeded into 10 cm dishes at 2 × 10⁶ cells dish⁻¹ 24 h before drug treatment. Six conditions in biological triplicate: (1) 250uM DMSO (vehicle for 250uM acyclovir), (2) 100nM DMSO (vehicle for 100nM branaplam), (3) acyclovir 250 uM, (4) branaplam 100 nM, (5) 500uM DMSO, (6) acyclovir 500 uM. Drugs or vehicle were added directly to culture medium; cells were incubated 72 h before harvest. Total RNA was isolated with TRIzol LS (Invitrogen 10296010) according to the manufacturer’s instructions. RNA pellets were resuspended in RNase-free H₂O, treated with 1 U DNase I (NEB M0303) 10 min at 37 °C, and purified with the RNA Clean & Concentrator-25 kit (Zymo R1018). RNA integrity (RIN > 9) was verified on an Agilent 2100 Bioanalyzer. RNA-seq libraries were prepared with the Illumina TruSeq Stranded Total RNA Library Preparation Kit (with rRNA depletion) at the Weill Cornell Medicine Genomics Resources Core Facility. Sequencing was performed on an Illumina NovaSeq X Plus platform, generating ≥70million 150 bp paired-end reads per sample. The 500 µM acyclovir group generated ≥ 32 million 150 bp paired-end reads per sample.