Fig. 5

How modifications increase knockout efficiency. a Knockout efficiency of sp3 from Fig. 2a with the indicated modifications was determined as in Fig. 1b. The raw data are shown in Figure S10 in Additional file 1. Mut mutant, O original. b sgRNA levels were determined by real-time PCR. The relative expression level was normalized to U6 small RNA. c In vitro transcribed sgRNA formed dimers (upper panel), which can be transformed into monomers by a heating and quick cooling step (lower panel). d sp7 from Fig. 3b was transcribed in vitro and preloaded into Cas9. The complex was electroporated into activated primary CD4+ T cells. Knockout efficiency was determined as in Fig. 3b. The raw data are shown in Figure S11 in Additional file 1. e In vitro transcribed sp7 was electroporated into TZM-Cas9 cells. Knockout efficiency was determined as in Fig. 3b. The raw data are shown in Figure S11 in Additional file 1. The graphs represent biological repeats from one of three independent experiments with similar results, shown as mean ± standard deviation (n = 3). Significance was calculated using Student's t-test: *P < 0.05; **P < 0.01