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

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

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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Crossref · 10.1038/s41467-018-03927-0

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  • Incorporation of bridged nucleic acids (2',4'-BNANC [N-Me]) or locked nucleic acids (LNA) into sgRNAs reduces the kinetics of Cas9 nuclease reactivity, thereby improving specificity of genome editing.

    “Incorporation of bridged nucleic acids (2’,4’-BNANC [N-Me]) or locked nucleic acids (LNA) into the sgRNAs also reduces the kinetics of Cas9 nuclease reactivity, thereby improving the specificity of genome editing”

    From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Cromwell et al., 2018

    The Abstract reports that BNANC-modified crRNAs slow Cas9 kinetics and improve specificity, and the Discussion links delayed cleavage to BNANC modification. The Results section on LNAs also reports improved specificity, but says Cas9 enzyme kinetics were reasonably similar with LNA-modified and unmodified crRNAs. The claimed kinetic reduction therefore does not hold for both modifications. (LLM, conf=0.89)

    Experimental — accuracy not yet measured
  • Chemical modifications on sgRNAs can influence their off-target effect.

    “Apart from sgRNA length adjustment, chemical modifications on sgRNAs can also influence their off-target effect.”

    From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Cromwell et al., 2018

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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.
Venue
Nature Communications
Published
2018-04-13
Volume
9
Issue
1
Pages
1448-1448
ISSN
2041-1723 (ISSN-L)
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
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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