The study investigates the off-target effects of RNA-guided endonucleases (RGENs) derived from the CRISPR/Cas system, which are used for targeted genome engineering. The authors demonstrate that by selecting unique target sequences and modifying the guide RNA and Cas9 protein, the off-target effects of RGENs can be reduced to below the detection limits of deep sequencing. They find that both the composition and structure of the guide RNA can affect RGEN activities, allowing them to discriminate on-target sites from off-target sites that differ by two bases. Exome sequencing analysis shows no off-target mutations induced by two RGENs in four clonal populations of mutant cells. Additionally, paired Cas9 nickases, which generate two single-strand breaks or nicks on different DNA strands, are highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. Paired nickases also induce chromosomal deletions in a targeted manner without causing unwanted translocations. The results highlight the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to minimize off-target mutations.The study investigates the off-target effects of RNA-guided endonucleases (RGENs) derived from the CRISPR/Cas system, which are used for targeted genome engineering. The authors demonstrate that by selecting unique target sequences and modifying the guide RNA and Cas9 protein, the off-target effects of RGENs can be reduced to below the detection limits of deep sequencing. They find that both the composition and structure of the guide RNA can affect RGEN activities, allowing them to discriminate on-target sites from off-target sites that differ by two bases. Exome sequencing analysis shows no off-target mutations induced by two RGENs in four clonal populations of mutant cells. Additionally, paired Cas9 nickases, which generate two single-strand breaks or nicks on different DNA strands, are highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. Paired nickases also induce chromosomal deletions in a targeted manner without causing unwanted translocations. The results highlight the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to minimize off-target mutations.