This study investigates the off-target effects of CRISPR/Cas-derived RNA-guided endonucleases (RGENs) and nickases in human cells. RGENs, derived from the prokaryotic CRISPR/Cas system, enable targeted genome editing by cleaving DNA at specific sites. However, they can cause off-target mutations and chromosomal translocations. The study shows that off-target effects can be significantly reduced by selecting unique target sequences and modifying guide RNA and Cas9. RGENs efficiently discriminate between on-target and off-target sites differing by two bases. Exome sequencing analysis revealed no off-target mutations in four clonal populations of mutant cells. Paired Cas9 nickases, which generate single-strand breaks (SSBs) on different DNA strands, were highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. These nickases induced targeted chromosomal deletions without unwanted translocations. The study highlights the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to minimize off-target effects. The results suggest that RGENs can distinguish on-target sites from those differing by at least two bases, and that careful selection of target sites can help avoid or reduce off-target mutations. The study also demonstrates that paired Cas9 nickases are more specific than traditional nucleases and can be used for precise genome editing. The findings contribute to the development of safer and more efficient genome editing tools.This study investigates the off-target effects of CRISPR/Cas-derived RNA-guided endonucleases (RGENs) and nickases in human cells. RGENs, derived from the prokaryotic CRISPR/Cas system, enable targeted genome editing by cleaving DNA at specific sites. However, they can cause off-target mutations and chromosomal translocations. The study shows that off-target effects can be significantly reduced by selecting unique target sequences and modifying guide RNA and Cas9. RGENs efficiently discriminate between on-target and off-target sites differing by two bases. Exome sequencing analysis revealed no off-target mutations in four clonal populations of mutant cells. Paired Cas9 nickases, which generate single-strand breaks (SSBs) on different DNA strands, were highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. These nickases induced targeted chromosomal deletions without unwanted translocations. The study highlights the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to minimize off-target effects. The results suggest that RGENs can distinguish on-target sites from those differing by at least two bases, and that careful selection of target sites can help avoid or reduce off-target mutations. The study also demonstrates that paired Cas9 nickases are more specific than traditional nucleases and can be used for precise genome editing. The findings contribute to the development of safer and more efficient genome editing tools.