PRJDB1494 Analyses of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. We previously addressed this issue by using the T7 endonuclease I (T7E1) assay and showed that RGENs do not induce mutations at potential off-target sites that are highly homologous to on-target sites in human cells(Cho et al. 2013a). But, due to its poor sensitivity, this assay cannot detect off-target mutations that occur at frequencies below 1%(Kim et al. 2009). Here, we address this critical issue more thoroughly by using deep sequencing, which can measure off-target mutations that occur at frequencies that range from 0.01 to 0.1%. In addition, we isolated clonal populations of cells that contained RGEN-induced mutations and investigated whether these mutant cells harbor any off-target mutations in the exome. Our results show that off-target effects of RGENs can be reduced in human cells by choosing unique target sequences and modifying the structure of crRNA or sgRNA. In addition, we report that paired Cas9 nickases that generate two single-strand breaks (SSBs) or nicks on opposite DNA strands are highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. National Creative Research Initiatives Center for Genome Engineering bioproject PRJDB1494 PRJDB1494 true pubmed 24253446