• Authors: Perez-Leal O. et al.
  • Year: 2021
  • Journal: CRISPR J
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell types:
    1. Name: HEK-293T
      Description: Human embryonic kidney Fibroblast
      Known as: HEK293T, 293T
    2. Name: HeLa
      Description: Human cervix epitheloid carcinoma cells


- Briefly, the Jetoptimus transfection reagent (Polyplus-transfection; Illkirch) was used in a reverse transfection reaction with endotoxin-free plasmids as indicated by the manufacturer. - The cells (1x10^4 in 100 µL per well) were seeded in triplicate on 384-well CellCarrier Ultra plates in FluoroBrite DMEM with 10% FBS and Glutamax. - The transfected cells were used for live-cell confocal imaging acquisition with an Operetta CLS Confocal High Content Imaging System with environmental control (37C and 5% CO2). For this, the culture media was exchanged 14 h after transfection, and images were acquired in four independent channels (mTagBFP2 [Ex405, Em-440], mClover3 [Ex-480, Em-513], mRuby3 [Ex-558, Em-590], and miRFP670 [Ex-642, Em-670]). - For HeLa cells, the images were captured every hour for 24 h by using a 40x water immersion objective. Four images (with four different channels) were acquired per well with the same coordinates in every well. - HeLa cells (2x10^5 cells per well) were transfected on 12- well plates with the plasmids encoding seven miRFP670- tagged SARS-CoV-2 proteins and a control miRFP670 plasmid. For this, we used the Jetoptimus transfection reagent as described above


The lack of efficient tools to label multiple endogenous targets in cell lines without staining or fixation has limited our ability to track physiological and pathological changes in cells over time via live-cell studies. Here, we outline the FAST-HDR vector system to be used in combination with CRISPR-Cas9 to allow visual live-cell studies of up to three endogenous proteins within the same cell line. Our approach utilizes a novel set of advanced donor plasmids for homology-directed repair and a streamlined workflow optimized for microscopy-based cell screening to create genetically modified cell lines that do not require staining or fixation to accommodate microscopy-based studies. We validated this new methodology by developing two advanced cell lines with three fluorescent-labeled endogenous proteins that support high-content imaging without using antibodies or exogenous staining. We applied this technology to study seven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/COVID-19) viral proteins to understand better their effects on autophagy, mitochondrial dynamics, and cell growth. Using these two cell lines, we were able to identify the protein ORF3a successfully as a potent inhibitor of autophagy, inducer of mitochondrial relocalization, and a growth inhibitor, which highlights the effectiveness of live-cell studies using this technology.