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

Cromwell, Christopher R; Sung, Keewon; Park, Jinho; Krysler, Amanda; Jovel, Juan; Kim, Seong Keun; Hubbard, Basil P.

doi:10.1038/s41467-018-03927-0

Cited by 168 publications

(168 citation statements)

References 50 publications

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“…The types of non-canonical gRNA are also continually expanding, including gRNA with DNA nucleotides 33 , chemical modifications 34,35 whose specificity improvement may also arise from DNA unwinding becoming more sensitive to mismatches. Non-canonical gRNAs can expand genome engineering applications a few additional ways.…”

Section: Discussionmentioning

confidence: 99%

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

Okafor

Singh

et al. 2019

Preprint

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Cas9 has made a wide range of genome engineering applications possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity but at the cost of the on-target activity. DNA unwinding is the primary checkpoint before cleavage by Cas9 and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in Cas9. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two guanines added were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5' end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.

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Section: Discussionmentioning

confidence: 99%

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

Okafor

Singh

et al. 2019

Preprint

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“…The underlying mechanism behind this change in specificity is unknown and intriguing given that an increase in GC content usually stabilises the hybridisation of RNA to DNA [51]. Finally, chemically modifying the central region of the 20-nucleotide guide sequence of the sgRNA through the inclusion of bridged or locked nucleic acids minimised OTEs [52]. The bridged/locked nucleic acids are thought to disrupt the stable state of the hybridised sgRNA-DNA complex at offtarget sites, hence impeding the formation of DSBs.…”

Section: Designing and Modifying The Sgrnamentioning

confidence: 99%

Mitigating off-target effects in CRISPR/Cas9-mediated in vivo gene editing

2020

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The rapid advancement of genome editing technologies has opened up new possibilities in the field of medicine. Nuclease-based techniques such as the CRISPR/Cas9 system are now used to target genetically linked disorders that were previously hard-totreat. The CRISPR/Cas9 gene editing approach wields several advantages over its contemporary editing systems, notably in the ease of component design, implementation and the option of multiplex genome editing. While results from the early phase clinical trials have been encouraging, the small patient population recruited into these trials hinders a conclusive assessment on the safety aspects of the CRISPR/Cas9 therapy. Potential safety concerns include the lack of fidelity in the CRISPR/Cas9 system which may lead to unintended DNA modifications at non-targeted gene loci. This review focuses modifications to the CRISPR/ Cas9 components that can mitigate off-target effects in in vitro and preclinical models and its translatability to gene therapy in patient populations.

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“…It tends to aggregate when consecutively modified on RNA and may have profound hepatoxicity. Cromwell et al [54] utilized a new generation bridged nucleic acid (BNA) analogue, 2 0 ,4 0 -BNA NC [N-Me], to optimize Cas9 sgRNA. It is believed to be more conformationally flexible and less toxic.…”

Section: Chemical Modificationmentioning

confidence: 99%

Engineering nucleic acid chemistry for precise and controllable CRISPR/Cas9 genome editing

Cai

Wang

2019

Science Bulletin

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

Cited by 168 publications

References 50 publications

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding

Mitigating off-target effects in CRISPR/Cas9-mediated in vivo gene editing

Engineering nucleic acid chemistry for precise and controllable CRISPR/Cas9 genome editing

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