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Cited by 371CRISPR and Genetic EngineeringVirus-based gene therapy researchRNA Interference and Gene Delivery

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

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Truong, Dong-Jiunn Jeffery, Kühner, Karin, Kühn, Ralf, Werfel, Stanislas, Engelhardt, Stefan, Wurst, Wolfgang, Ortiz, Oskar·2015·Nucleic Acids Research
DOI 10.1093/nar/gkv601PMC PMC4513872
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Abstract

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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  • An alternate method for expressing large proteins relying on intein protein sequences has been reported.

    “An alternate method for expressing large proteins relying on intein protein sequences has also been reported (Li et al., 2008; Truong et al., 2015; Chew et al., 2016).”

    From Evaluating Efficiencies of Dual AAV Approaches for Retinal Targeting (2017) · cites this paper as Truong et al., 2015

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Authors
Truong, D.-J. J., Kühner, K., Kühn, R., Werfel, S., Engelhardt, S., Wurst, W., & Ortiz, O.
Affiliations
  • Helmholtz Munich (DE)
  • Max Delbrück Center (DE)
  • Technical University of Munich (DE)
  • German Centre for Cardiovascular Research (DE)
  • German Center for Neurodegenerative Diseases (DE)
Corresponding author
  • Wurst, Wolfgang
  • Ortiz, Oskar
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2015-06-16
Volume
43
Issue
13
Pages
6450-6458
ISSN
  • 0305-1048 (ISSN-L)
  • 1362-4962
DOI
10.1093/nar/gkv601
PMC
PMC4513872
OpenAlex
W1636931877
Cited by
371
References
5
Citations by year
2026: 17 · 2025: 32 · 2024: 36 · 2023: 43 · 2022: 49 · 2021: 49 · 2020: 37 · 2019: 26 · 2018: 25 · 2017: 33 · 2016: 17 · 2015: 7
FWCI
11.87
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • MDC Repository (Max-Delbrueck-Center for Molecular Medicine) (acceptedVersion)
  • Open access LMU (Ludwid Maxmilian's Universitat Munchen) (submittedVersion)
  • mediaTUM (Technical University of Munich) (submittedVersion)
  • Site cant be reached (submittedVersion, cc-by)
  • DZNE Pub (submittedVersion, cc-by)
  • PuSH - Publication Server of Helmholtz Zentrum München (submittedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
split intein, CRISPR/Cas9, gene therapy, recombinant adeno-associated virus, Cas9 nickase, transient transfection, genome editing
MeSH terms
Dependovirus, Cell Line, Deoxyribonucleases, Humans, Plasmids, Streptococcus pyogenes, Transfection, Genetic Therapy, Gene Targeting, Inteins, CRISPR-Cas Systems, CRISPR-Associated Proteins

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