T7 endonuclease Assay

T7 endonuclease I assay to detect genomic modifications

U2OS-EGFP cells or HEK293T cells were transfected with Cas9 expression and sgRNA expression plasmids or linear DNA PCR products as described above or treated with only Cas9 protein, only in vitro transcribed sgRNA, or only RNAiMAX. Genomic DNA was isolated from cells 2 days after transfection using the DNAdvance Kit (Agencourt) following the manufacturer’s instructions. 200 ng of genomic DNA was used as template in PCR reactions to amplify the targeted genomic loci with flanking survey primer pairs specified in the Supplementary Notes. PCR products were purified with a QIAquick PCR Purification Kit (Qiagen) and quantified with Quant-iT™ PicoGreen ® dsDNA Kit (Life Technologies). 250ng of purified PCR DNA was combined with 2 µL of NEBuffer 2 (NEB) in a total volume of 19 µL and denatured then re-annealed with thermocycling at 95 °C for 5 min, 95 to 85 °C at 2 °C/s; 85 to 20 °C at 0.2 °C/s. The re-annealed DNA was incubated with 1 µl of T7 Endonuclease I (10 U/µl, NEB) at 37 °C for 15 min. 10 µL of 50 % glycerol was added to the T7 Endonuclease reaction and 12 µL was analyzed on a 5 % TBE 18-well Criterion PAGE gel (Bio-Rad) electrophoresed for 30 min at 200 V, then stained with 1x SYBR Gold (Life Technologies) for 30 min. Cas9-induced cleavage bands and the uncleaved band were visualized on an AlphaImager HP (Alpha Innotech) and quantified using ImageJ software48. The peak intensities of the cleaved bands were divided by the total intensity of all bands (uncleaved + cleaved bands) to determine the fraction cleaved which was used to estimate gene modification levels as previously described.40 For each sample, transfections and subsequent modification measurements were performed in triplicate on different days.