CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences

By Yanni Lin, Thomas J. Cradick, Matthew T. Brown, Harshavardhan Deshmukh, P. Ranjan, Neha Sarode, B. Wile, P. Vertino, Frank Stewart, Gang Bao. Nucleic Acids Research, 2014

620 citations recorded by the source corpus.

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.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/8850a542d8f6f55c99f24f13513c1829e47dae53

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Citations in the corpus

The CRISPR/Cas9 system for plant genome editing and beyond. (2015, background): “DNA sequences that contain an extra base (DNA bulge) or a missing base (gRNA bulge) at various locations along the corresponding gRNA sequence have also been shown to induce off-target cleavage (Lin et al., 2014).”
Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 (2016, positive): “Asymmetric sgRNA:DNA interactions that lead to bulged RNA or DNA bases have been examined for a small number of examples and our results are qualitatively consistent 41 .”
Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites (2015, background): “However, RGENs can also cleave off-target DNA sites with several mismatches (Cho et al., 2014; Fu et al., 2013) or with missing or additional nucleotides (Lin et al., 2014), compared to gRNA sequences.”
Target specificity of the CRISPR-Cas9 system (2014, neutral): “that contain insertions or deletions relative to the guide RNA; however many of these sites could be alternatively aligned to contain only mismatches to the guide [61].”
The potential for the use of gene drives for pest control in New Zealand: a perspective (2018, background): “It should also be noted that CRISPR/Cas9 targeting events also cause off-target effects, causing sequence change distant from the targeted site (Lin et al. 2014; Shin et al. 2017).”
Enhanced Rice Blast Resistance by CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor Gene OsERF922 (2016, result): “For example, previous studies have demonstrated that off-target effects were common at the level of one nucleotide mismatch in plant species [37, 50], fish [79] and human cells [80, 81].”
CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites (2014, neutral): “Conflict of interest: none declared.”
The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells (2016, positive): “Previous studies have shown that Spy CRISPR-Cas9 systems can tolerate genomic target sites containing base mismatches, single-base DNA insertions (DNA bulges) and single-base DNA deletions (RNA bulges), 13–16 which have significant implications to the targeting specificity of CRISPR-Cas9.”