Figure 4.

Knockout of Venus transgene by inducing indels using gRNA-encoding plasmids. MEF cells carrying a single-copy of the Venus transgene were electrotransfected with different gRNA-encoding plasmids (9.1 kb length). Induction of indels using gRNA-72 had no effect on the Venus expression, whereas gRNA + 100 resulted in loss of the Venus signal. Electroporated cells were selected against puromycin and screened 10 days after the electroporation. (a and b) Cells were stained with Hoechst 33342 and the efficiency of Venus knockout was assessed. Scale bar equals 10 µm. (c) Histoplots of the FACS results. (d) Partial sequencing results of the amplified Venus transgene. Target sites of the respective gRNAs are indicated by a red line, the first three nucleotides of the electropherogram are cropped to fit the image into the column. (e) Indels spectrum calculated by online tool for TIDE analysis. Cells with no copy of Venus were considered as the negative control (NK), and cells carrying a copy of Venus, without any electroporation treatment, were considered as the positive control for the Venus expression (PK). The following electroporation program was used: square-wave protocol: 20 µg plasmid (1.5–2.5 µg/µl), 300 V, 2 pulses, each 10 ms pulse length, 10 s pulse interval, and 4 mm cuvette (n > 3).