293T, HepG2,
and PANC1 cells cultivated in the 24-well plates were cotransfected
with 400 ng of TALEN vectors (200 ng left TALEN and 200 ng right TALEN)
and 400 ng of homology arm donor. 293T, HepG2, and PANC1 cells cultivated
in the 24-well plates were cotransfected with 400 ng of sgRNA-CRISPR/Cas9
and 400 ng of homology arm donor. The cells were cultured for an additional
24 h. When the donor AviTag-IRES2-displayAviTag was used, the cells
were cotransfected with an additional 100 ng pMy-BirA (Addgene).
One microliter of the IRDye800CW-conjugated streptavidin (1 mg/mL
stock; Li-COR Bioscience) was added to the cells in the wells and
cultivated for 4 h. The cells were rinsed with PBS and imaged using
the Odyssey Infrared Imaging System (Li-COR Bioscience) at the channel
of NIRF 800 nm. One microliter of the Alexa Fluor 488-conjugated Streptavidin
(0.9 mg/mL stock; YEASEN, China) was added to the cells in wells and
cultivated overnight. The cells were rinsed with PBS and photographed
using a fluorescence microscope, IX51 with DP71 (Olympus), and quantitatively
analyzed using a flow cytometer, Calibur (BD). At this point, the
efficiency of gene editing was also determined by estimating the fraction
of the fluorescent cells using a fluorescence microscope and ImageJ
counting.
and PANC1 cells cultivated in the 24-well plates were cotransfected
with 400 ng of TALEN vectors (200 ng left TALEN and 200 ng right TALEN)
and 400 ng of homology arm donor. 293T, HepG2, and PANC1 cells cultivated
in the 24-well plates were cotransfected with 400 ng of sgRNA-CRISPR/Cas9
and 400 ng of homology arm donor. The cells were cultured for an additional
24 h. When the donor AviTag-IRES2-displayAviTag was used, the cells
were cotransfected with an additional 100 ng pMy-BirA (Addgene).
One microliter of the IRDye800CW-conjugated streptavidin (1 mg/mL
stock; Li-COR Bioscience) was added to the cells in the wells and
cultivated for 4 h. The cells were rinsed with PBS and imaged using
the Odyssey Infrared Imaging System (Li-COR Bioscience) at the channel
of NIRF 800 nm. One microliter of the Alexa Fluor 488-conjugated Streptavidin
(0.9 mg/mL stock; YEASEN, China) was added to the cells in wells and
cultivated overnight. The cells were rinsed with PBS and photographed
using a fluorescence microscope, IX51 with DP71 (Olympus), and quantitatively
analyzed using a flow cytometer, Calibur (BD). At this point, the
efficiency of gene editing was also determined by estimating the fraction
of the fluorescent cells using a fluorescence microscope and ImageJ
counting.