Protocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells.

Authors

Marina A Tyumentseva, Aleksandr I Tyumentsev, Vasiliy G Akimkin

Published in

PloS one. Volume 16. Issue 11. Pages e0259812. Epub Nov 09, 2021.

Abstract

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 methods that will not affect viability of target cell dramatically. Popular current methods and protocols of CRISPR/Cas RNP delivery include lipofection and electroporation, transduction by osmocytosis and reversible permeabilization and erythrocyte-based 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.
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.

PMID:
34752487
Bibliographic data and abstract were imported from PubMed on 07 Nov 2025.

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