CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences.
Bottom Line: A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large mammalian genomes.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.