title: Development of an intein-mediated split-Cas9 system for gene therapy.
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.
authors.affiliation: Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich 85764, Germany Institute of Developmental Genetics,Technische Universität München, Freising-Weihenstephan 85354, Germany.
authors.forename: Dong-Jiunn Jeffery
authors.initials: DJ
authors.lastname: Truong
authors.affiliation: Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich 85764, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Kühner
authors.affiliation: Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich 85764, Germany Max Delbrück Center for Molecular Medicine (MDC), Berlin 13125, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Kühn
authors.affiliation: Institute of Pharmacology and Toxicology. Technische Universität München, Munich 80802, Germany.
authors.forename: Stanislas
authors.initials: S
authors.lastname: Werfel
authors.affiliation: Institute of Pharmacology and Toxicology. Technische Universität München, Munich 80802, Germany German Center for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, Munich 80802, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Engelhardt
authors.affiliation: Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich 85764, Germany Institute of Developmental Genetics,Technische Universität München, Freising-Weihenstephan 85354, Germany Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich 80336, Germany Munich Cluster for Systems Neurology (SyNergy) Adolf-Butenandt-Institut Ludwig-Maximilians-Universität München, Munich 81377, Germany oskar.ortiz@helmholtz-muenchen.de.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wurst
authors.affiliation: Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Munich 85764, Germany oskar.ortiz@helmholtz-muenchen.de.
authors.forename: Oskar
authors.initials: O
authors.lastname: Ortiz
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics*; D064113:CRISPR-Cas Systems; D002460:Cell Line; D003851:Deoxyribonucleases / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics; D018390:Gene Targeting; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D047668:Inteins; D010957:Plasmids / Q000235:genetics; D013297:Streptococcus pyogenes / Q000201:enzymology; D014162:Transfection
chemical_list: D064130:CRISPR-Associated Proteins; D003851:Deoxyribonucleases