CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences.
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.
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Yanni
Y
Lin
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Thomas J
TJ
Cradick
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Matthew T
MT
Brown
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Harshavardhan
H
Deshmukh
School of Biology, Georgia Institute of Technology, Atlanta, GA 30332, USA.
Piyush
P
Ranjan
School of Biology, Georgia Institute of Technology, Atlanta, GA 30332, USA.
Neha
N
Sarode
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Brian M
BM
Wile
Department of Radiation Oncology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Paula M
PM
Vertino
School of Biology, Georgia Institute of Technology, Atlanta, GA 30332, USA.
Frank J
FJ
Stewart
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA gang.bao@bme.gatech.edu.
Gang
G
Bao
D001482:Base Composition; D020137:Base Pair Mismatch; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D003596:Cytosine / Q000032:analysis; D004247:DNA / Q000737:chemistry; D053837:DNA Cleavage; D003851:Deoxyribonucleases / Q000378:metabolism*; D006147:Guanine / Q000032:analysis; D057809:HEK293 Cells; D006801:Humans; D017384:Sequence Deletion; D058727:RNA, Small Untranslated
D064130:CRISPR-Associated Proteins; D006147:Guanine; D003596:Cytosine; D004247:DNA; D003851:Deoxyribonucleases; D058727:RNA, Small Untranslated