CRISPR/Cas9 systems are a versatile tool for genome editing due to the highly 
efficient targeting of DNA sequences complementary to their RNA guide strands. 
However, it has been shown that RNA-guided Cas9 nuclease cleaves genomic DNA 
sequences containing mismatches to the guide strand. A better understanding of 
the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large 
mammalian genomes. Here we show that genomic sites could be cleaved by 
CRISPR/Cas9 systems when DNA sequences contain insertions ('DNA bulge') or 
deletions ('RNA bulge') compared to the RNA guide strand, and Cas9 nickases used 
for paired nicking can also tolerate bulges in one of the guide strands. 
Variants of single-guide RNAs (sgRNAs) for four endogenous loci were used as 
model systems, and their cleavage activities were quantified at different 
positions with 1- to 5-bp bulges. We further investigated 114 putative genomic 
off-target loci of 27 different sgRNAs and confirmed 15 off-target sites, each 
harboring a single-base bulge and one to three mismatches to the guide strand. 
Our results strongly indicate the need to perform comprehensive off-target 
analysis related to DNA and sgRNA bulges in addition to base mismatches, and 
suggest specific guidelines for reducing potential off-target cleavage.