Citation
- Authors: Hatch ST. et al.
- Year: 2022
- Journal: Methods S1046-2023 00148-7
- Applications: in vitro / DNA / jetOPTIMUS
- Cell type: Lenti-X 293T
Method
Culture HEK293xT cells (632180, Takara Bio) in D10 media (DMEM, 11965–092, Gibco; 100 U/mL Penicillin-Streptomycin, 15140122, Gibco; 10% FBS, 26140079, Gibco) at 37 °C/5% CO2. At 50–80% density, dry trypsinize (aspirate media, wash with trypsin, then aspirate and transfer to incubator for 30–60 s until cells are spherical in morphology) cells using TrypLE (12604013, Gibco). Dilute cells to 2.5 × 10^5 cells/mL, adding 300 uL/well to a clear 48-well plate (3548, Corning).
When 48-well cultures reach 60–80% density, prepare for transfection by warming jetOPTIMUS DNA transfection reagent (76299–632, VWR) to room temperature. For each well, combine 30 uL jetOPTIMUS buffer and 0.3 uL jetOPTIMUS reagent, vortex for 5 s, then dilute 150 ng plasmid DNA in the buffer/reagent mix, vortex 5 s, then spin. Incubate at room temperature for 10 min.
Controls include a positive transfection control, pmaxGFP (sold as part of Lonza nucleofection kits), an “empty” vector control under a CMV promoter (V79520, ThermoFisher Scientific), and a non-targeting control (NT/NTC). Guide design/plasmid/cloning methods outlined in this work were used to generate a NTC U1 snRNA with a 25 nucleotide (nt) guide sequence containing no significant similarity to the human genome (NCBI BLAST, Supplementary Table 3).
Add 30 uL/well of the corresponding transfection mix dropwise, not allowing the pipette tip to touch the media upon addition. Gently orbitally shake the plate by hand, and place in incubator for a ∼ 48 h transfection period.
After ∼48 h post-transfection, confirm 60–80% transfection efficiency via GFP-based transfection controls.
Abstract
Alternative splicing accounts for a considerable portion of transcriptomic diversity, as most protein-coding genes are spliced into multiple mRNA isoforms. However, errors in splicing patterns can give rise to mis-splicing with pathological consequences, such as the congenital diseases familial dysautonomia, Duchenne muscular dystrophy, and spinal muscular atrophy. Small nuclear RNA (snRNA) components of the U snRNP family have been proposed as a therapeutic modality for the treatment of mis-splicing. U1 snRNAs offer great promise, with prior studies demonstrating in vivo efficacy, suggesting additional preclinical development is merited. Improvements in enabling technologies, including screening methodologies, gene delivery vectors, and relevant considerations from gene editing approaches justify further advancement of U1 snRNA as a therapeutic and research tool. With the goal of providing a user-friendly protocol, we compile and demonstrate a methodological toolkit for sequence-specific targeted perturbation of alternatively spliced pre-mRNA with engineered U1 snRNAs. We observe robust modulation of endogenous pre-mRNA transcripts targeted in two contrasting splicing contexts, SMN2 exon 7 and FAS exon 6, exhibiting the utility and applicability of engineered U1 snRNA to both inclusion and exclusion of targeted exons. We anticipate that these demonstrations will contribute to the usability of U1 snRNA in investigating splicing modulation in eukaryotic cells, increasing accessibility to the broader research community.