Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System
- Qazi, Shefah [ Montana State University: Microbiology & Cell Biology ] [ Department of Chemistry, Indiana University ]
- Miettinen, Heini M. [ Montana State University: Microbiology & Cell Biology ]
- McCoy, Kimberly [ Department of Chemistry, Indiana University ]
- Douglas, Trevor [ Montana State University: Microbiology & Cell Biology ] [ Department of Chemistry, Indiana University ]
- Wiedenheft, Blake [ Montana State University: Microbiology & Cell Biology ]
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.