Background
Delivery of CRISPR/Cas RNPs to target cells still remains the biggest bottleneck to genome editing. Many efforts are made to develop efficient CRISPR/Cas RNP delivery
Discussion
This will be the first extensive comparative study of popular current methods and protocols of CRISPR/Cas RNP delivery to human cell lines and primary cells. All protocols will be optimized and characterized using the following criteria i) protein delivery and genome editing efficacy; ii) viability of target cells after delivery (post-transduction recovery); iii) scalability of delivery process; iv) cost-effectiveness of the delivery process and v) intellectual property rights. Some methods will be considered 'research-use only', others will be recommended for scaling and application in the development of cell-based therapies.
Methods
In this study we will assess the efficiency and optimize current CRISPR/Cas RNP delivery protocols to target cells. We will conduct our work using molecular cloning, protein expression and purification, cell culture, flow cytometry (immunocytochemistry) and cellular imaging techniques.