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Fig. 1 | Genome Biology

Fig. 1

From: Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects

Fig. 1

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. β€œCon” 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 β€œMethods” section

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