Fig. 1
CRISPR/Cas9 efficiently generates single DSBs and DSB clusters at the hHPRT locus. (A) Schematic representation of Exon 3 of the hHPRT gene showing the target sites of the guide RNAs (gRNAs) utilized. The distances between DSBs are indicated in base pairs (bp). The nucleotide sequences of the gRNAs are provided in the lower part. The flanking 5’-nucleotides, as well as the corresponding PAM sequences are indicated. (B) Representative images of 53BP1 foci after co-transfection of A549 cells with Cas9-GFP and the indicated combinations of gRNA expression vectors. Only GFP positive cells are monitored in these experiments. (C) Fold increase in mutation frequency (MF) in A549 cells transfected with gRNA expression vectors inducing single DSBs at the indicated locations in and around Exon 3 of hHPRT. Data represents means and standard deviations (SD) from two biological repeats. Individual data points from each determination are also shown. (D) Fold increase of MF in A549 cells, 3 days after transfection with combinations of gRNA expression vectors inducing DSB clusters of increasing complexity, i.e. increasing number of DSBs. The background MF for the selected experiments was between 1.05 × 10− 5 and 1.86 × 10− 5 (Table S2). The bar plot represents the mean and standard deviation (SD) from 4 to 6 independent experiments. Dots indicate the individual values determined in each experiment. The corresponding P-values are shown in Table S1.
