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RNA-guided retargeting of Sleeping Beauty transposition in human cells

Authors: Adrian Kovač; Csaba Miskey; Michael Menzel; Esther Grueso; Andreas Gogol-Döring; Zoltán Ivics;

RNA-guided retargeting of Sleeping Beauty transposition in human cells

Abstract

An ideal tool for gene therapy would enable efficient gene integration at predetermined sites in the human genome. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, and occurred in an asymmetric pattern with a bias towards sites in a relatively narrow, 300 bp window downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.

Country
Spain
Keywords

Hypoxanthine Phosphoribosyltransferase, Retroelements, QH301-705.5, Science, Gentherapie ; Transposon, Transposases, RNA, Guide, CRISPR-Cas Systems, gene targeting, DNA-binding, CRISPR/Cas, Biochemistry and Chemical Biology, Humans, Biology (General), genetic engineering, Chromosomes, Human, X, Genome, Human, Q, R, Reproducibility of Results, Genetic Therapy, Multigene Family, Medicine, CRISPR-Cas Systems, gene insertion, HeLa Cells

  • BIP!
    Impact byBIP!
    selected citations
    These citations are derived from selected sources.
    This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
    39
    popularity
    This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
    Top 10%
    influence
    This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
    Top 10%
    impulse
    This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
    Top 10%
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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
39
Top 10%
Top 10%
Top 10%
Green
gold