CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences.
Bottom Line: 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.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.
Affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.Show MeSH
Mentions: We further investigated if sgRNAs with small truncations at the 5′-end retain cleavage activity. One to six nucleotides were deleted from the 5′ end of R-01 except for the nucleotide at position 20, because the guanine here is required for the expression under the U6 promoter (Figure 3A). For these guide sequence truncations, we found that 1- to 2-bp 5′ truncations could still induce cleavage activities similar to the full-length sgRNA (Figure 3B).
Affiliation: Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.