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Cited by 166

The DOI matches its Crossref record; 13 of the 13 references it cites were found in a registry or an index.

Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system

Liu, Xiaoxi, Homma, Ayaka, Sayadi, Jamasb, Yang, Shu, Ohashi, Jun, Takumi, Toru·2016·Scientific Reports
DOI 10.1038/srep19675PMC PMC4728555
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Abstract

The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications.

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Crossref · 10.1038/srep19675

Cites

13 of 13 references verified0 citation errors · 0 not checkable

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  • Mismatches in the seed sequence terminate pairing and compromise Cas9 cleaving activity, while mismatches towards the 5′ PAM-distal end do not always jeopardise Cas9 function.

    “While mismatches in the seed sequence terminate pairing and compromise Cas9 cleaving activity, mismatches towards the 5′ PAM-distal end do not always jeopardise Cas9 function [67].”

    From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [67]

    The Abstract (page unavailable) says PAM-proximal seed bases are important for targeting specificity and that PAM-distal mismatches are more likely to be tolerated. The Discussion (page unavailable) also links seed-region positions to cleavage efficiency. Neither passage says seed mismatches terminate pairing, so that mechanistic part of the claim is unsupported. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

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Citations

Cites

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Research works printed but not linked: 24

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Metadata

Authors
Liu, X., Homma, A., Sayadi, J., Yang, S., Ohashi, J., & Takumi, T.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2016-01-27
Volume
6
Issue
1
ISSN
2045-2322
DOI
10.1038/srep19675
PMC
PMC4728555
Cited by
166
References
13
Language
en
Licence

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