Genome-wide, unbiased off-target analysis was performed following the iGUIDE pipeline based on Guide-seq invented previously (62 (link),63 (link)). HEK293T cells were transfected in 20 uL Lonza SF Cell Line Nucleofector Solution on a Lonza Nucleofector 4D with program DS-150 according to the manufacturer's instructions. 300 ng of gRNA–Cas9 plasmids (or 150 ng of each gRNA–Cas9n plasmid for the double nickase), 150 ng of the effector plasmids, and 5 pmol of double stranded oligonucleotides (dsODN) were transfected. Cells were harvested after 72 h for genomic DNA using Agencourt DNAdvance reagent kit. 400 ng of purified gDNA which was then fragmented to an average of 500 bp and ligated with adaptors using NEBNext Ultra II FS DNA Library Prep kit following manufacturer's instructions. Two rounds of nested anchored PCR from the oligo tag to the ligated adaptor sequence were performed to amplify targeted DNA, and the amplified library was purified, size-selected and sequenced using Illumina Miseq V2 PE300 or V3 PE600. Sequencing data was analyzed using the published iGUIDE pipeline, with the addition of a downsampling step which ensures an unbiased comparison across samples.