RNA-guided retargeting of S leeping Beauty transposition in human cells

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 (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to i...

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Bibliographic Details
Published ineLife Vol. 9
Main Authors Kovač, Adrian, Miskey, Csaba, Menzel, Michael, Grueso, Esther, Gogol-Döring, Andreas, Ivics, Zoltán
Format Journal Article
LanguageEnglish
Published England 06.03.2020
Subjects
Chromosomes, Human, X
CRISPR-Cas Systems
Genetic Therapy
Genome, Human
HeLa Cells
Humans
Hypoxanthine Phosphoribosyltransferase
Multigene Family
Reproducibility of Results
Retroelements
RNA, Guide, CRISPR-Cas Systems - genetics
RNA, Guide, CRISPR-Cas Systems - metabolism
Transposases - genetics
Transposases - metabolism
chemical biology
gene insertion
biochemistry
gene targeting
CRISPR/Cas
human
DNA-binding
genetic engineering
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Summary: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 (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 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.
ISSN:2050-084X