RNA-guided endonucleases (RGENs), derived from the prokaryotic adaptive immune 
system known as CRISPR/Cas, enable targeted genome engineering in cells and 
organisms. RGENs are ribonucleoproteins that consist of guide RNA and Cas9, a 
protein component originated from Streptococcus pyogenes. These enzymes cleave 
chromosomal DNA, whose sequence is complementary, to guide RNA in a targeted 
manner, producing site-specific DNA double-strand breaks (DSBs), the repair of 
which gives rise to targeted genome modifications. Despite broad interest in 
RGEN-mediated genome editing, these nucleases are limited by off-target 
mutations and unwanted chromosomal translocations associated with off-target DNA 
cleavages. Here, we show that off-target effects of RGENs can be reduced below 
the detection limits of deep sequencing by choosing unique target sequences in 
the genome and modifying both guide RNA and Cas9. We found that both the 
composition and structure of guide RNA can affect RGEN activities in cells to 
reduce off-target effects. RGENs efficiently discriminated on-target sites from 
off-target sites that differ by two bases. Furthermore, exome sequencing 
analysis showed that no off-target mutations were induced by two RGENs in four 
clonal populations of mutant cells. In addition, paired Cas9 nickases, composed 
of D10A Cas9 and guide RNA, which generate two single-strand breaks (SSBs) or 
nicks on different DNA strands, were highly specific in human cells, avoiding 
off-target mutations without sacrificing genome-editing efficiency. 
Interestingly, paired nickases induced chromosomal deletions in a targeted 
manner without causing unwanted translocations. Our results highlight the 
importance of choosing unique target sequences and optimizing guide RNA and Cas9 
to avoid or reduce RGEN-induced off-target mutations.