Manufacturing and Delivering Genome-Editing Proteins.

Area of Science:

• Biomedical Sciences
• Molecular Biology
• Gene Editing Technologies

Background:

• Genome-editing technologies offer powerful tools for genetic modification.
• Efficient and precise delivery of gene-editing tools is crucial for minimizing unintended alterations.
• Purified protein delivery, such as Cas9 ribonucleoproteins (RNPs), is an effective strategy to reduce off-target mutations.

Purpose of the Study:

• To provide detailed protocols for the manufacturing and delivery of genome-modifying proteins into mammalian cells.
• To describe methods for using Cas9 RNPs with adeno-associated virus (AAV) donor vectors.
• To enable seamless transgene insertion via homology-directed genome editing.

Main Methods:

• Step-by-step procedures for producing and delivering genome-modifying proteins, including Cas9 RNPs, TALE nucleases, and zinc-finger nucleases.
• Protocols for preparing and introducing purified protein complexes into mammalian cells.
• Methods for combining Cas9 RNPs with adeno-associated virus (AAV) donor vectors for homology-directed repair.

Main Results:

• Demonstrated effective delivery of genome-modifying proteins into mammalian cells.
• Established protocols for generating and utilizing Cas9 RNPs for gene editing.
• Successfully integrated protocols for combining Cas9 RNPs with AAV vectors for precise transgene insertion.

Conclusions:

• The described methods facilitate efficient and targeted genome modification in mammalian cells.
• Purified protein delivery offers a robust approach for precise gene editing applications.
• These protocols support advanced genome engineering, including seamless transgene integration using Cas9 RNPs and AAV vectors.