CRISPR-Cas9 Genome Editing in HEK 293T

For the genome-editing, the DNA-directed RNA-guided endonuclease (RGEN) system (TakaraBio) was used [20 (link)]. Designed gRNA sequence was integrated into the expression vector under the U6 promotor (pRGEN_U6_SG). Cas9 endonuclease was integrated into the expression vector under the CMV promotor (pRGEN-Cas9-CMV). These plasmids were transfected into E.coli and purified by the NucleoBond Xtra Midi EF (Macherey-Nagel). The 71–78 bp long single-stranded oligonucleotide (ssODN) (100 pmol) was transfected along pRGEN_U6_SG (0.17 μg) and pRGEN-Cas9-CMV (0.25 μg) vectors into 1.75 × 10⁵ HEK 293 T/17 cells by using TransIT-X2 reagent (Mirus Bio). Then the cells were cultured in 24 well tissue culture plates for 3 days. As an alternative method for 4 genes (AKT3, BIM, IGF2 and MYCN), gRNA was prepared by in vitro transcription (IVT) using Guide-it sgRNA In Vitro Transcription Kit (Takara) and was transfected with Cas9 protein by the Neon Transfection System (Thermo Fisher Scientific) with two pulses of 1100 V and 20 ms.
Regarding the method of introducing the Cas9, a transfection of the Cas9 proteins as a complex with gRNA (RNP) was used in the later experiment [20 (link)], instead of the standard expression vector method.