Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Cellular Delivery Systems

Background:

• Efficient delivery of CRISPR-Cas9 ribonucleoprotein complexes (Cas9 RNPs) into mammalian cells is crucial for gene editing applications.
• Optimizing transfection reagents and conditions is essential for maximizing genome modification efficiency.

Purpose of the Study:

• To identify the optimal lipid nanoparticles for delivering Cas9 RNPs into mammalian cells.
• To establish the best transfection conditions for efficient gene editing.

Main Methods:

• Systematic screening of 60 transfection reagents across six common mammalian cell lines.
• Optimization of transfection parameters to enhance delivery and editing efficiency.
• Evaluation of genome editing efficiencies in various cell types, including HEK293FT, mouse ES cells, and human iPSCs.

Main Results:

• A novel reagent, Lipofectamine CRISPRMAX, was identified as superior to existing methods.
• Lipofectamine CRISPRMAX demonstrated 40% and 15% higher genome modification efficiencies compared to Lipofectamine 3000 and RNAiMAX, respectively.
• Optimized transfection achieved high genome editing efficiencies: 85% in HEK293FT cells, 75% in mouse ES cells, and 55% in human iPSCs.
• Successful co-delivery of donor DNA with Cas9 RNPs resulted in up to 17% EmGFP-positive cells.

Conclusions:

• Lipofectamine CRISPRMAX is the leading lipid nanoparticle formulation for Cas9 RNP delivery into diverse mammalian cell lines.
• The optimized protocol enables efficient gene editing in critical cell types like mouse embryonic stem cells and induced pluripotent stem cells.