title: High Efficiency CRISPR/Cas9-mediated Gene Editing in Primary Human T-cells Using Mutant Adenoviral E4orf6/E1b55k "Helper" Proteins.
abstract: Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.
authors.affiliation: Program for Cell and Gene Therapy, Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington, USA.
authors.forename: Kamila S
authors.initials: KS
authors.lastname: Gwiazda
authors.affiliation: Program for Cell and Gene Therapy, Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington, USA.|Department of Immunology, University of Washington, Seattle, Washington, USA.
authors.forename: Alexandra E
authors.initials: AE
authors.lastname: Grier
authors.affiliation: Program for Cell and Gene Therapy, Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington, USA.
authors.forename: Jaya
authors.initials: J
authors.lastname: Sahni
authors.affiliation: Program for Cell and Gene Therapy, Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington, USA.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Burleigh
authors.affiliation: Program for Cell and Gene Therapy, Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington, USA.
authors.forename: Unja
authors.initials: U
authors.lastname: Martin
authors.affiliation: Program for Cell and Gene Therapy, Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington, USA.
authors.forename: Julia G
authors.initials: JG
authors.lastname: Yang
authors.affiliation: Program for Cell and Gene Therapy, Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington, USA.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Popp
authors.affiliation: Program for Cell and Gene Therapy, Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington, USA.
authors.forename: Michelle C
authors.initials: MC
authors.lastname: Krutein
authors.affiliation: Program for Cell and Gene Therapy, Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington, USA.
authors.forename: Iram F
authors.initials: IF
authors.lastname: Khan
authors.affiliation: Program for Cell and Gene Therapy, Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington, USA.
authors.forename: Kyle
authors.initials: K
authors.lastname: Jacoby
authors.affiliation: Ben Towne Center for Childhood Cancer Research, Seattle Children's Research Institute, Seattle, Washington, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jensen
authors.affiliation: Program for Cell and Gene Therapy, Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington, USA.|Department of Immunology, University of Washington, Seattle, Washington, USA.|Department of Pediatrics, University of Washington, Washington, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Rawlings
authors.affiliation: Program for Cell and Gene Therapy, Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington, USA.|Department of Immunology, University of Washington, Seattle, Washington, USA.|Department of Pediatrics, University of Washington, Washington, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scharenberg
mesh_terms: D064113:CRISPR-Cas Systems; D000229:Dependovirus / Q000235:genetics; D000072669:Gene Editing; D015870:Gene Expression; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D023061:Gene Order; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D059765:Homologous Recombination; D006801:Humans; D050505:Mutant Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D013601:T-Lymphocytes / Q000378:metabolism*; D014161:Transduction, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C553319:E1B55K protein, adenovirus; D050505:Mutant Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D014764:Viral Proteins