title: Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System.
abstract: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA-guided endonucleases are powerful new tools for targeted genome engineering. These nucleases provide an efficient and precise method for manipulating eukaryotic genomes; however, delivery of these reagents to specific cell-types remains challenging. Virus-like particles (VLPs) derived from bacteriophage P22, are robust supramolecular protein cage structures with demonstrated utility for cell type-specific delivery of encapsulated cargos. Here, we genetically fuse Cas9 to a truncated form of the P22 scaffold protein, which acts as a template for capsid assembly as well as a specific encapsulation signal for Cas9. Our results indicate that Cas9 and a single-guide RNA are packaged inside the P22 VLP, and activity assays indicate that this RNA-guided endonuclease is functional for sequence-specific cleavage of dsDNA targets. This work demonstrates the potential for developing P22 as a delivery vehicle for cell specific targeting of Cas9.
authors.affiliation: Department of Microbiology and Immunology, Montana State University , Bozeman, Montana 59717, United States.|Department of Chemistry, Indiana University , 800 East Kirkwood Avenue, Bloomington, Indiana 47405, United States.
authors.forename: Shefah
authors.initials: S
authors.lastname: Qazi
authors.affiliation: Department of Microbiology and Immunology, Montana State University , Bozeman, Montana 59717, United States.
authors.forename: Heini M
authors.initials: HM
authors.lastname: Miettinen
authors.affiliation: Department of Microbiology and Immunology, Montana State University , Bozeman, Montana 59717, United States.
authors.forename: Royce A
authors.initials: RA
authors.lastname: Wilkinson
authors.affiliation: Department of Chemistry, Indiana University , 800 East Kirkwood Avenue, Bloomington, Indiana 47405, United States.
authors.forename: Kimberly
authors.initials: K
authors.lastname: McCoy
authors.affiliation: Department of Chemistry, Indiana University , 800 East Kirkwood Avenue, Bloomington, Indiana 47405, United States.
authors.forename: Trevor
authors.initials: T
authors.lastname: Douglas
authors.affiliation: Department of Microbiology and Immunology, Montana State University , Bozeman, Montana 59717, United States.
authors.forename: Blake
authors.initials: B
authors.lastname: Wiedenheft
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D017100:Bacteriophage P22 / Q000378:metabolism*; D000076987:CRISPR-Associated Protein 9; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004337:Drug Carriers / Q000737:chemistry*; D016503:Drug Delivery Systems; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D053758:Nanoparticles / Q000737:chemistry*; D017393:RNA Editing / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004337:Drug Carriers; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: Cas9; P22 bacteriophage; VLP; delivery vehicle; gene therapy; genome editing; virus-like particles