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Truong, D.J.J. ; Kühner, K. ; Kühn, R. ; Werfel, S.* ; Engelhardt, S.* ; Wurst, W. ; Ortiz, O.

Development of an intein-mediated split-Cas9 system for gene therapy.

Nucleic Acids Res. 43, 6450-6458 (2015)
Publ. Version/Full Text DOI PMC
Open Access Gold
Creative Commons Lizenzvertrag
Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV.
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Publication type Article: Journal article
Document type Scientific Article
Corresponding Author
Keywords Mouse Embryos; Stem-cells; Dual-rna; Cas9; Activation; Transcription; Expression; Immunity; Dna; Crispr/cas9
ISSN (print) / ISBN 0305-1048
e-ISSN 1362-4962
Journal Nucleic Acids Research
Quellenangaben Volume: 43, Issue: 13, Pages: 6450-6458 Article Number: , Supplement: ,
Publisher Oxford University Press
Publishing Place Oxford
Non-patent literature Publications
Reviewing status Peer reviewed
Institute(s) Institute of Developmental Genetics (IDG)