Snippet: Virus-like particles (VLPs) were generated in HEK293T cells via co-transfection of pBS‑CMV‑gagpol (RRID: Addgene_35614), pCMV‑MMLVgag‑3xNES‑Cas9 (RRID: Addgene_181752), pCMV‑VSV‑G (RRID: Addgene_8454), and the pU6‑sgRNA plasmid using an MLV‑based production system30 . HEK293T cells were plated in T150 flasks and transfected at 70–80% confluence using TurboFectin 8.0 (Origene, #TF81001)

Snippet: Trypan blue, 0.04% Thermo Fisher Scientific Cat#15250061 0.25% trypsin-EDTA Gibco Cat#25200-056 TurboFectin 8.0 Transfection Reagent OriGene SKU TF81001

Snippet: pCMV-VSV-G (Addgene #8454), and the transfer vectors pHIV-H2B-eGFP (Addgene #91776) or pHIV-H2B-mRFP (Addgene #18982). Transductions were performed using Turbofectin 8.0 (OriGene, TF81001). Viral supernatants were harvested at 48 and 72 hours post-transfection, filtered, pooled, and stored at -80°C

Snippet: ng opto-RhoA, geneticin was added to the medium at a concentration of 0.4 mg/mL. Cell culture flasks were maintained at 37°C in a 5% CO2 environment. Cells were passaged every 2 − 3 day when they reached 60-80% confluency. The transgenic HeLa cell line was generated through lipofection using Turbofectin 8.0 (#TF81001, OriGene) according to the manufacturer’