title: Baculovirus-based genome editing in primary cells.
abstract: Genome editing in eukaryotes became easier in the last years with the development of nucleases that induce double strand breaks in DNA at user-defined sites. CRISPR/Cas9-based genome editing is currently one of the most powerful strategies. In the easiest case, a nuclease (e.g. Cas9) and a target defining guide RNA (gRNA) are transferred into a target cell. Non-homologous end joining (NHEJ) repair of the DNA break following Cas9 cleavage can lead to inactivation of the target gene. Specific repair or insertion of DNA with Homology Directed Repair (HDR) needs the simultaneous delivery of a repair template. Recombinant Lentivirus or Adenovirus genomes have enough capacity for a nuclease coding sequence and the gRNA but are usually too small to also carry large targeting constructs. We recently showed that a baculovirus-based multigene expression system (MultiPrime) can be used for genome editing in primary cells since it possesses the necessary capacity to carry the nuclease and gRNA expression constructs and the HDR targeting sequences. Here we present new Acceptor plasmids for MultiPrime that allow simplified cloning of baculoviruses for genome editing and we show their functionality in primary cells with limited life span and induced pluripotent stem cells (iPS).
authors.affiliation: Paul Scherrer Institute, Biomolecular Research, Molecular Cell Biology, CH-5232 Villigen, Switzerland; ETH Zürich, Department of Biology, CH-8093 Zürich, Switzerland.
authors.forename: Maysam
authors.initials: M
authors.lastname: Mansouri
authors.affiliation: University of Basel, Department of Biomedicine, CH-4058 Basel, Switzerland.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Ehsaei
authors.affiliation: University of Basel, Department of Biomedicine, CH-4058 Basel, Switzerland.
authors.forename: Verdon
authors.initials: V
authors.lastname: Taylor
authors.affiliation: Paul Scherrer Institute, Biomolecular Research, Molecular Cell Biology, CH-5232 Villigen, Switzerland. Electronic address: Philipp.Berger@psi.ch.
authors.forename: Philipp
authors.initials: P
authors.lastname: Berger
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D064113:CRISPR-Cas Systems; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004720:Endonucleases / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D025741:HMGA1a Protein / Q000235:genetics* / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism / Q000821:virology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism / Q000821:virology; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D025741:HMGA1a Protein; D004720:Endonucleases
keywords: Baculovirus; CRISPR/Cas9; Genome editing; Primary cells