Figure 2.
Nuclease comparison in Jurkat T-ALL cell line. Nucleic acids were delivered by electroporation into Jurkats and 7 days later, the amount of CD3 loss from the cell surface was determined by flow cytometry. (a) The first lane is the GFP transfection control with 95+% CD3 expression levels. Lane two shows the MT disruption rates. Lanes three and four are TALEN optimization conditions. TALEN mRNA was generated from a T3 polymerase promoter without (T3 alone) or with (T3 + pA) a exogenous polyadenylation signal. (b) Cas9 mRNA and protein with varying platforms of gRNA-editing rates. The first lane is GFP followed by a linear DNA fragment encoding the gRNA (Cas9, linear) or circularized gRNA plasmid (Cas9, plasmid) borne expression systems. Following that are a gRNA transcript (Cas9, RNA) produced locally by in vitro transcription and commercially synthesized gRNAs that are unmodified or modified with 2′O-Methyl (2′-OMe) bases that were delivered at doses of 5 or 10 μg by either complexing with Cas9 protein (RNP) or with Cas9 mRNA. Statistical comparisons were done using the Student's t-test. *,***, and **** represent P values (Student's t-test) of <0.05, <0.001, and <0.0001, respectively. Data are from four independent experiments.