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Cited by 190CRISPR and Genetic EngineeringDNA and Nucleic Acid ChemistryAdvanced biosensing and bioanalysis techniques

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Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity

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Cromwell, Christopher R., Sung, Keewon, Park, Jinho, Krysler, Amanda R., Jovel, Juan, Kim, Seong Keun, Hubbard, Basil P.·2018·Nature Communications
DOI 10.1038/s41467-018-03927-0PMC PMC5899152
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Abstract

Abstract Off-target DNA cleavage is a paramount concern when applying CRISPR-Cas9 gene-editing technology to functional genetics and human therapeutic applications. Here, we show that incorporation of next-generation bridged nucleic acids (2′,4′-BNA NC [N-Me]) as well as locked nucleic acids (LNA) at specific locations in CRISPR-RNAs (crRNAs) broadly reduces off-target DNA cleavage by Cas9 in vitro and in cells by several orders of magnitude. Using single-molecule FRET experiments we show that BNA NC incorporation slows Cas9 kinetics and improves specificity by inducing a highly dynamic crRNA–DNA duplex for off-target sequences, which shortens dwell time in the cleavage-competent, “zipped” conformation. In addition to describing a robust technique for improving the precision of CRISPR/Cas9-based gene editing, this study illuminates an application of synthetic nucleic acids.

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Figures

Fig. 1 BNANC incorporation reduces off-target cleavage in vitro. a Chemical structures of RNA, LNA (2′,4′-BNA), and BNANC (2′,4′-BNANC [NMe]) nucleotides. b WAS and c EMX1 on-target and off-target sequences used for in vitro and cellular cleavage assays. Mismatches are indicated by red lowercase lettering. Heat map showing in vitro cleavage specificity for the unmodified crRNA and 9 BNANC-modified crRNAs toward either d WAS or e EMX1 on-target and off-target sequences (as listed in Fig. 1b, c); mean shown (n = 2). crRNA and BNANC-modified sequences are shown to the left of the corresponding heat map. BNANC modifications are indicated in black. Targets that were highly cleaved in vitro are indicated in red, while targets that were not cleaved are indicated in blue. Gel showing relative cleavage efficiencies of the unmodified and most specific BNANC-modified crRNAs on a 1-kb DNA fragment containing either the f WAS or g EMX1 on-target and off-target sequences. The two bottom bands are cleavage products, while the top band is full-length substrate. The molar ratio of Cas9 RNP complex to target DNA was 30:1 for these experiments. Quantification of cleavage percentages was determined using densitometry (ImageJ), and are shown below each lane. Lanes in which no cleavage products were observed are marked as undetected (UD). Values used to generate heatmaps are presented in Supplementary Table 1
Fig. 1 BNANC incorporation reduces off-target cleavage in vitro. a Chemical structures of RNA, LNA (2′,4′-BNA), and BNANC (2′,4′-BNANC [NMe]) nucleotides. b WAS and c EMX1 on-target and off-target sequences used for in vitro and cellular cleavage assays. Mismatches are indicated by red lowercase lettering. Heat map showing in vitro cleavage specificity for the unmodified crRNA and 9 BNANC-modified crRNAs toward either d WAS or e EMX1 on-target and off-target sequences (as listed in Fig. 1b, c); mean shown (n = 2). crRNA and BNANC-modified sequences are shown to the left of the corresponding heat map. BNANC modifications are indicated in black. Targets that were highly cleaved in vitro are indicated in red, while targets that were not cleaved are indicated in blue. Gel showing relative cleavage efficiencies of the unmodified and most specific BNANC-modified crRNAs on a 1-kb DNA fragment containing either the f WAS or g EMX1 on-target and off-target sequences. The two bottom bands are cleavage products, while the top band is full-length substrate. The molar ratio of Cas9 RNP complex to target DNA was 30:1 for these experiments. Quantification of cleavage percentages was determined using densitometry (ImageJ), and are shown below each lane. Lanes in which no cleavage products were observed are marked as undetected (UD). Values used to generate heatmaps are presented in Supplementary Table 1CC BY · PubMed Central
Fig. 2 BNANC incorporation broadly improves specificity in vitro. Heat maps showing DNA cleavage specificity scores across >1012 off-target sequences for either unmodified (top) or BNANC-modified crRNAs (bottom) targeting a WAS or b EMX1. Specificity scores of 1.0 (dark blue) correspond to 100% enrichment for, while scores of −1.0 (dark red) correspond to 100% enrichment against a specific base-pair at a specific position. Black boxes denote the intended target nucleotides. Bar graph showing the quantitative difference in specificity score at each position in the 20 base-pair target site and 2 base-pair PAM (N of NGG excluded), between the unmodified and BNANC-modified crRNA for c WAS or d EMX1 target sequences. A score of zero indicates no change in specificity. Difference in specificity was calculated as specificity scoreBNANC−specificity scoreRNA. Experiments were performed with 200 nM pre-selection library and 1000 nM Cas9 RNP complex
Fig. 2 BNANC incorporation broadly improves specificity in vitro. Heat maps showing DNA cleavage specificity scores across >1012 off-target sequences for either unmodified (top) or BNANC-modified crRNAs (bottom) targeting a WAS or b EMX1. Specificity scores of 1.0 (dark blue) correspond to 100% enrichment for, while scores of −1.0 (dark red) correspond to 100% enrichment against a specific base-pair at a specific position. Black boxes denote the intended target nucleotides. Bar graph showing the quantitative difference in specificity score at each position in the 20 base-pair target site and 2 base-pair PAM (N of NGG excluded), between the unmodified and BNANC-modified crRNA for c WAS or d EMX1 target sequences. A score of zero indicates no change in specificity. Difference in specificity was calculated as specificity scoreBNANC−specificity scoreRNA. Experiments were performed with 200 nM pre-selection library and 1000 nM Cas9 RNP complexCC BY · PubMed Central
Fig. 3 BNANC incorporation increases Cas9 specificity in cells. Gel showing relative cellular cleavage efficiencies of the unmodified, and 9 BNANC-modified crRNAs targeting a WAS or b EMX1 on-target (top) or off-target (bottom) sequences, as determined by T7 endonuclease I digestion. Mock transfections lacking guide RNA were used as controls. Modification frequencies were determined using densitometry (ImageJ) and are indicated below each lane. Lanes in which no cleavage products were observed are marked as undetected (UD)
Fig. 3 BNANC incorporation increases Cas9 specificity in cells. Gel showing relative cellular cleavage efficiencies of the unmodified, and 9 BNANC-modified crRNAs targeting a WAS or b EMX1 on-target (top) or off-target (bottom) sequences, as determined by T7 endonuclease I digestion. Mock transfections lacking guide RNA were used as controls. Modification frequencies were determined using densitometry (ImageJ) and are indicated below each lane. Lanes in which no cleavage products were observed are marked as undetected (UD)CC BY · PubMed Central
Fig. 4 BNANC incorporation influences conformational transitions. a Schematic diagram for smFRET experiments showing a Cas9 RNP complex consisting of Cy5-labeled crRNA, tracrRNA, and Cas9, bound to a Cy3-labeled dsDNA immobilized on a quartz surface. b Histograms showing FRET efficiency after equilibration for the WAS DNA (upper) or WAS-OT4 DNA (lower) target sequences using WAS-RNA (dark blue) or WAS-BNA-3 (light blue) crRNAs; black curves represent Gaussian fits. c Time trace for Cas9 on WAS-OT4 DNA using WAS-BNA-3 crRNA indicating repetitive transitions between the open and zipped conformations. Dwell time in each conformation is indicated as ∆τ. d Comparison of Cas9 dwell times between WAS-RNA and WAS-BNA-3 crRNA using the WAS-OT4 DNA template; mean ± SD shown
Fig. 4 BNANC incorporation influences conformational transitions. a Schematic diagram for smFRET experiments showing a Cas9 RNP complex consisting of Cy5-labeled crRNA, tracrRNA, and Cas9, bound to a Cy3-labeled dsDNA immobilized on a quartz surface. b Histograms showing FRET efficiency after equilibration for the WAS DNA (upper) or WAS-OT4 DNA (lower) target sequences using WAS-RNA (dark blue) or WAS-BNA-3 (light blue) crRNAs; black curves represent Gaussian fits. c Time trace for Cas9 on WAS-OT4 DNA using WAS-BNA-3 crRNA indicating repetitive transitions between the open and zipped conformations. Dwell time in each conformation is indicated as ∆τ. d Comparison of Cas9 dwell times between WAS-RNA and WAS-BNA-3 crRNA using the WAS-OT4 DNA template; mean ± SD shownCC BY · PubMed Central

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Metadata

Authors
Cromwell, C. R., Sung, K., Park, J., Krysler, A. R., Jovel, J., Kim, S. K., & Hubbard, B. P.
Affiliations
  • University of Alberta (CA)
  • Seoul National University (KR)
Corresponding author
Hubbard, Basil P.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2018-04-13
Volume
9
Issue
1
Pages
1448-1448
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-018-03927-0
PMC
PMC5899152
OpenAlex
W2796961959
Cited by
190
References
50
Citations by year
2026: 13 · 2025: 15 · 2024: 29 · 2023: 19 · 2022: 30 · 2021: 33 · 2020: 26 · 2019: 17 · 2018: 8
FWCI
7.47
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Universities Space Research Association
  • National Research Foundation
  • National Research Foundation of Korea (2018R1A2B2001422)
  • Natural Sciences and Engineering Research Council of Canada (rgpin-2016-06381, RGPIN-2016)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
Keywords
CRISPR/Cas9, bridged nucleic acids, locked nucleic acid, single-molecule FRET, dwell time, synthetic nucleic acids
MeSH terms
RNA, Guide, CRISPR-Cas Systems, DNA, Endonucleases, Escherichia coli, HeLa Cells, Humans, RNA, Sensitivity and Specificity, Streptococcus pyogenes, RNA, Guide, Kinetoplastida, Polymorphism, Single Nucleotide, Fluorescence Resonance Energy Transfer, Cell Line, Tumor, DNA Cleavage, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems

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