Gc buffer

DNA was isolated with the QiaAMP DNA Mini Kit (Qiagen) at 3 days post transfection of vectors encoding the respective CRISPR nucleases. The potentially disrupted locus was amplified using locus specific primers and PRECISOR polymerase in GC-buffer (BioCat). PCR products were purified using either the QiaQuick gel extraction kit (for PCR products including unspecific products) or the QiaQuick purification kit. Heterodimerization and digestion with SURVEYOR nuclease were performed with the SURVEYOR Mutation Detection Kit (Transgenomic) according to the manufacturer’s instructions. The cleavage products were separated on a 2% agarose gel and stained with ethidium bromide for 10 min. Images were captured with the Gel Doc system (Bio-Rad). Band intensities were quantified via ImageJ. Gene modification levels were calculated using the following equation^{19 (link)}: $$\%\mathrm{genes}\mathrm{modified}=(1-{(1-\mathrm{fraction}\mathrm{cleaved})}^{0.5})\times 100$$

A total of 2 × 10⁵ NIH3T3 cells (obtained from ATCC, authenticated as murine and mycoplasma free) were seeded in six-well dishes containing 2 ml IMDM medium (Life Technologies; supplemented with FCS, penicillin/streptavidin and glutamine) per well 1 day before transfection. Lipofectamine 2,000 (Life Technologies) was used to transfect cells with 3 μg Cas9/gRNA expression vector according to the manufacturer's instructions. DNA was isolated with the QiaAMP DNA Mini Kit (Qiagen) at 3 days post transfection. The respective locus was amplified using the primers Survey_PTCH_fwd 5′-GGATGGTCCTGGTTCCAGTC-3′ and Survey500_PTCH_rev 5′-CCCGAGTAGATTACAGCGCG-3′ with PRECISOR polymerase (Biocat) in GC buffer (Biocat). Heterodimerization and digestion with SURVEYOR nuclease were performed with the SURVEYOR Mutation Detection Kit (Transgenomic) according to the manufacturer's instructions. Cleavage products were separated on a 2% agarose gel and stained with ethidium bromide for 10 min. Images were captured with the Gel Doc system (Bio-Rad). Band intensities were quantified via ImageJ. Gene modification levels were calculated using the following equation44 (link): % genes modified=(1−(1−fraction cleaved)^0.5 ) × 100.