In vitro gene editing using Cas9 mRNA. (A) IVT Cas9 mRNA was generated with modified nucleotides, N1-Methylpseudouridine (m1Ψ), and transfected into 293T-EGFP cells with two types of EGFP-targeting gRNA. EGFP expression in non-treated (NC) and transfected cells was observed by microscopy (100X) (B) and quantified using flow cytometry (C). (D) At 48 or 72 h post transfection, the EGFP expression was reduced to about 40% of the non-treated 293T-EGFP cells. (E and F) The editing to EGFP genes on the genome was further confirmed by T7E1 assay (E) and Sanger sequencing (F). The highlighted adenine indicates the gene editing introduced an insertion in the sequence.