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  Refined sgRNA efficacy prediction improves large- and small-scale CRISPR -Cas9 applications

Labuhn, M., Adams, F. F., Ng, M., Knoess, S., Schambach, A., Charpentier, E. M., et al. (2018). Refined sgRNA efficacy prediction improves large- and small-scale CRISPR -Cas9 applications. Nucleic Acids Research (London), 46(3), 1375-1385. doi:10.1093/nar/gkx1268.

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Labuhn, Maurice, Autor
Adams, Felix F., Autor
Ng, Michelle, Autor
Knoess, Sabine, Autor
Schambach, Axel, Autor
Charpentier, Emmanuelle M.1, Autor
Schwarzer, Adrian, Autor
Mateo, Juan L., Autor
Klusmann, Jan-Henning, Autor
Heckl, Dirk, Autor
Affiliations:
1Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Max Planck Society, Charitéplatz 1, D-10117 Berlin, DE, ou_2266691              

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Schlagwörter: Algorithms, Base Composition, Base Sequence, Cell Line, Tumor, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Gene Editing, Gene Targeting, HEK293 Cells, Humans, Leukocytes, Nucleic Acid Conformation, RNA, Guide
 Zusammenfassung: Genome editing with the CRISPR-Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, the effort to eliminate variability in sgRNA efficacies-which affect experimental sensitivity-is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verification at selected endogenous loci, we experimentally quantified the target efficacies of 430 sgRNAs. Based on this dataset we tested the predictive value of five recently-established prediction algorithms. Our analysis revealed a moderate correlation (r = 0.04 to r = 0.20) between the predicted and measured activity of the sgRNAs, and modest concordance between the different algorithms. We uncovered a strong PAM-distal GC-content-dependent activity, which enabled the exclusion of inactive sgRNAs. By deriving nine additional predictive features we generated a linear model-based discrete system for the efficient selection (r = 0.4) of effective sgRNAs (CRISPRater). We proved our algorithms' efficacy on small and large external datasets, and provide a versatile combined on- and off-target sgRNA scanning platform. Altogether, our study highlights current issues and efforts in sgRNA efficacy prediction, and provides an easily-applicable discrete system for selecting efficient sgRNAs.

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 Datum: 2018-02
 Publikationsstatus: Erschienen
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 Identifikatoren: DOI: 10.1093/nar/gkx1268
BibTex Citekey: labuhn_refined_2018
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Titel: Nucleic Acids Research (London)
  Kurztitel : Nucleic Acids Res
Genre der Quelle: Zeitschrift
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Ort, Verlag, Ausgabe: Oxford : Oxford University Press
Seiten: - Band / Heft: 46 (3) Artikelnummer: - Start- / Endseite: 1375 - 1385 Identifikator: ISSN: 0305-1048
CoNE: https://pure.mpg.de/cone/journals/resource/110992357379342