Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR RNA-guided endonucleases offer precise genome engineering but face delivery challenges.
• Virus-like particles (VLPs) from bacteriophage P22 are effective for targeted cargo delivery.
• Efficient delivery of genome engineering tools to specific cell types is crucial.

Purpose of the Study:

• To develop a novel delivery system for CRISPR-Cas9 using P22 VLPs.
• To demonstrate the packaging and functionality of Cas9 within P22 VLPs.
• To explore the potential of P22 VLPs for cell-specific CRISPR-Cas9 delivery.

Main Methods:

• Genetically fusing the Cas9 enzyme to a truncated P22 scaffold protein.
• Assembling P22 virus-like particles (VLPs) with encapsulated Cas9 and guide RNA.
• Performing activity assays to confirm sequence-specific DNA cleavage by packaged Cas9.

Main Results:

• Cas9 and single-guide RNA were successfully packaged into P22 VLPs.
• The encapsulated Cas9 demonstrated functional RNA-guided endonuclease activity.
• Sequence-specific cleavage of double-stranded DNA (dsDNA) targets was confirmed.

Conclusions:

• P22 VLPs can serve as a viable vehicle for delivering functional CRISPR-Cas9.
• This method shows promise for cell-specific genome engineering applications.
• Further development of P22 VLPs could enhance targeted delivery of gene editing tools.