Fig. 4

Dual Cas9 nickase strategy mediates DNA DSBs with single stranded overhangs. A 6 different guides were used for the dual Cas9 nickase strategy. Three guides are complement to the sense strand (Guide A1, A2, and A3) and three guides target the anti-sense strand (Guide B1, B2, and B3). Two different Cas9 nickases were applied, Cas9 D10A and Cas9 H840A. B Two Cas9 D10A utilizing guides at a tail-to-tail orientation, will generate a DNA DSB with 5ʹ overhang ssDNA. As for C dual Cas9 H840A strategy will generate a 3ʹ overhang DNA DSB