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.
© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
Schematic of CRISPR/Cas9 off-target sites with (
A) 1-bp insertion (DNA bulge) or ( B) 1-bp deletion (RNA bulge). The 20-nt guide sequence (orange line) in the sgRNA is shown with genomic target sequence (protospacer) containing single-base DNA bulge (red asterisk) or single-base sgRNA bulge (red Δ). The zoom-in sequences of protospacer and PAM are shown above the sgRNA guide sequence. Positions of nucleotides in the target are numbered 3′ to 5′ starting from the nucleotide next to PAM.
Activity of sgRNA variants targeted to genomic loci containing single-base DNA bulges. A single nucleotide was deleted from the original sgRNA at all possible positions (red dashes) throughout the guide sequence for (
A) sgRNA R-01 targeting HBB or ( B) sgRNA R-30 targeting CCR5. Cleavage activity for the corresponding sgRNA variants measured by T7E1 assay in HEK293T cells at ( C) the HBB site or ( D) CCR5 site for the sgRNA variants in (A) and (B). Sequence of the original sgRNA is in the top row of the grid. Positions of the deleted nucleotides are highlighted for A (green), G (black), C (blue), or U (red) in the grid. Semi-transparent colors in two positions in the same sgRNA indicate that deletions can be interpreted at either of adjacent positions (also marked by ‘or’) due to identical nucleotides at both positions. The bar graph on the right shows cleavage activity aligned to the corresponding sgRNA variants using the same color scheme. Positions relative to PAM are labeled on the y-axis. The vertical dashed lines mark the activity levels of the original sgRNAs. Error bar, SEM ( n = 2).
Activity for sgRNAs containing 5′-end truncations. (
A) 1–6 bp truncations at the 5′ end of the guide sequence R-01 targeted to the HBB gene. ( B) Activity for truncated sgRNAs. Truncated positions are highlighted in gray in the grid. Bar graph shows corresponding cleavage activity measured by T7E1 assay in HEK293T cells. Error bar, SEM ( n = 2).
Activity of sgRNA variants targeted to genomic loci containing single-base sgRNA bulges. (A and B) Activity of Cas9 at
(A) HBB target site and (B) CCR5 target site carrying single-base sgRNA bulges associated with different variants of the original sgRNAs (A) R-01 and (B) R-30. Single nucleotide, A (green), G (black), C (blue), or U (red), was inserted into the original sgRNA throughout the guide sequence. Sequence of the original sgRNA is in the top row of the grid. Positions of the original guide sequence are shaded in gray, while the inserted positions are white. Due to identical nucleotides at adjacent positions, some inserted nucleotides can be in multiple positions (marked by ‘or’). Bar graphs on the right show corresponding cleavage activities quantified by T7E1 assay in HEK293T cells, with the same color scheme for different inserted nucleotides. Positions relative to PAM and the single nucleotides added are labeled on the y-axis. Error bar, SEM ( n = 2).
Activity of sgRNA variants with bulges targeted to genomic loci with different GC contents. (
A) Target sites, cleavage activities (% indels by T7E1 assay) and GC contents of different guide strands targeted to HBB and CCR5 genes. *Cleavage activity of R-25 is from reference (22). (B and C) T7E1 activity of R-08 variants targeted to HBB genomic loci with ( B) single-base DNA bulges or ( C) single-base sgRNA bulges. Color schemes and labels are similar to Figures 2 and 4. Error bar, SEM ( n = 2).
Activity of sgRNA variants with 2-bp DNA or 2- to 5-bp sgRNA bulges. Guide strands with 2- to 5-bp addition are labeled with ‘+’ and positions of the added bases and guide strands with 2-bp deletion are labeled with ‘−’ and positions of the deleted bases. (
A) Sequences comparison of guide RNAs and target sites, with position numbers on top. ( B) Bar graph showing cleavage activities of these sgRNA variants quantified by T7E1 assay in HEK293T cells. Error bar, SEM ( n = 2).
Paired Cas9 nickases with one bulge-containing sgRNA effectively cleave genomic DNA. (
A) Human HBB gene targeted by Cas9 nickases (Cas9n) with paired guide strands R-01 and R-02. PAMs are indicated with grey bars. ( B) T7E1 activities of Cas9n with R-01 bulge-variants paired with R-02, compared with original Cas9 activities of the R-01 bulge-variants as in Figures 2 and 4. Error bar, SEM ( n = 2). Asterisks indicate P-values from a two-tailed independent two-sample t-test. * P < 0.05, ** P < 0.01, *** P < 0.001.
Activities of CRISPR/Cas9 nucleases at genomic target sites and at off-target sites with single-base DNA bulges coupled with mismatches. (A and B) On-target and off-target cleavage activities for (
A) sgRNAs R-30 targeted to CCR5 gene, and ( B) R-31 target to ERCC5 gene. Upper: target sequences ( CCR5 and ERCC5) and off-target sequences (Off-4 and Off-1) with mismatch (red) and DNA bulge (shaded in yellow) shown next to the sgRNA (R-30 and R-31) tested. Red lines indicate the PAM. Bottom: Cleavage activities at the target sites and off-target sites measured by T7E1 assay in HEK293T cells. ‘−’ and ‘+’ denote samples treated without and with nuclease, respectively. Numbers below the lanes indicate average percentages of gene modification ( n = 2). Asterisks indicate specific T7E1 cleavage products. (C and D) Sanger sequencing reads of amplified off-target sites aligned to the wild-type genomic sequence and sgRNAs for ( C) R-30 and ( D) R-31. The occurrence of each sequence is indicated to the left of the alignment, if greater than one. Unmodified reads are indicated by ‘WT’. Deletions are marked in gray and insertions marked in yellow. (E) Significant activities analyzed by deep sequencing at genomic off-target loci containing bulges coupled with mismatches and in some cases alternative NAG-PAM. Only bulge-containing off-target loci determined to have P-values less than 0.05 are shown. Table on the left shows numbers of mismatches at off-target loci in addition to bulge (no. of mis), bulge types, positions of bulges from PAM (bulge pos), labels for the loci as in Supplementary Table S6 and sequences of off-target sites including PAMs. In these off-target genomic sequences, mismatches are marked by red, deleted base compared to sgRNA marked as ‘−’ (sgRNA bulge), inserted base compared to sgRNA marked as underlined red letters (DNA bulge), NAG-PAMs are marked by blue. Bar graph on the right indicates indel percentages quantified for mock (blue) and treated samples (red) with sgRNAs at off-target loci shown in the table to the left. Error bars, Wilson intervals (see ‘Materials and Methods’ section). * P ≤ 0.05, *** P ≤ 0.001 as determined by Fisher's exact test. The % indel values of treated samples are also indicated.
All figures (8)
Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases.
Genome Res. 2014 Jan;24(1):132-41. doi: 10.1101/gr.162339.113. Epub 2013 Nov 19.
Genome Res. 2014.
24253446 Free PMC article.
CRISPRseek: a bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems.
PLoS One. 2014 Sep 23;9(9):e108424. doi: 10.1371/journal.pone.0108424. eCollection 2014.
PLoS One. 2014.
25247697 Free PMC article.
CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity.
Nucleic Acids Res. 2013 Nov;41(20):9584-92. doi: 10.1093/nar/gkt714. Epub 2013 Aug 11.
Nucleic Acids Res. 2013.
23939622 Free PMC article.
CRISPR-Cas9 Structures and Mechanisms.
Annu Rev Biophys. 2017 May 22;46:505-529. doi: 10.1146/annurev-biophys-062215-010822. Epub 2017 Mar 30.
Annu Rev Biophys. 2017.
Potential pitfalls of CRISPR/Cas9-mediated genome editing.
FEBS J. 2016 Apr;283(7):1218-31. doi: 10.1111/febs.13586. Epub 2015 Nov 27.
FEBS J. 2016.
Mitigating off-target effects in CRISPR/Cas9-mediated in vivo gene editing.
J Mol Med (Berl). 2020 May;98(5):615-632. doi: 10.1007/s00109-020-01893-z. Epub 2020 Mar 20.
J Mol Med (Berl). 2020.
32198625 Free PMC article.
CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off-Target Evaluation, and Strategies to Mitigate Off-Target Effects.
Adv Sci (Weinh). 2020 Feb 6;7(6):1902312. doi: 10.1002/advs.201902312. eCollection 2020 Mar.
Adv Sci (Weinh). 2020.
32195078 Free PMC article.
Allele-specific genome targeting in the development of precision medicine.
Theranostics. 2020 Feb 10;10(7):3118-3137. doi: 10.7150/thno.43298. eCollection 2020.
32194858 Free PMC article.
OffScan: a universal and fast CRISPR off-target sites detection tool.
BMC Genomics. 2020 Mar 5;21(Suppl 1):872. doi: 10.1186/s12864-019-6241-9.
BMC Genomics. 2020.
32138651 Free PMC article.
Bolotin A., Quinquis B., Sorokin A., Ehrlich S.D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology. 2005;151:2551–2561.
Horvath P., Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010;327:167–170.
Marraffini L.A., Sontheimer E.J. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat. Rev. Genet. 2010;11:181–190.
Garneau J.E., Dupuis M., Villion M., Romero D.A., Barrangou R., Boyaval P., Fremaux C., Horvath P., Magadán A.H., Moineau S. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 2010;468:67–71.
Hale C.R., Zhao P., Olson S., Duff M.O., Graveley B.R., Wells L., Terns R.M., Terns M.P. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell. 2009;139:945–956.
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