Fig. 1
From: Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects
F-A locus and target site selection. a The molecular structures of the human, mouse, and cattle F-A loci were highly conserved. b Schematic representation of the plasmid encoding both hSpCas9 expression and sgRNA transcription. Different sgRNAs were cloned for the corresponding target sites through the two neighboring BbsI restriction enzyme sites. c Concentration-dependent cleavage activity and the amount of pSpCas9 DNA transfected. The three potential off-target sites were selected with Cas-OFFinder based on fewer than three mismatches and the NHEJ frequency was determined by a Surveyor nuclease assay. d Cleavage efficiencies of the Cas9 protein at the selected target sites. “Con” represents the DNA isolated from BFFs transfected with Cas9 only (without sgRNA). The degree of cleavage was quantified with Surveyor nuclease assays and ImageJ (http://imagej.net). The formula for estimating the indel rate is provided in the “Methods” section