Protocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells
<h4>Background</h4> 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 proto...
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oai:doaj.org-article:29be6d3838f34a0492e637a62af0d06e2021-11-18T06:34:24ZProtocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells1932-6203https://doaj.org/article/29be6d3838f34a0492e637a62af0d06e2021-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8577758/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4> 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. <h4>Methods</h4> 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. <h4>Discussion</h4> 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.Marina A. TyumentsevaAleksandr I. TyumentsevVasiliy G. AkimkinPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 11 (2021) |
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Medicine R Science Q Marina A. Tyumentseva Aleksandr I. Tyumentsev Vasiliy G. Akimkin Protocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells |
description |
<h4>Background</h4> 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. <h4>Methods</h4> 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. <h4>Discussion</h4> 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. |
format |
article |
author |
Marina A. Tyumentseva Aleksandr I. Tyumentsev Vasiliy G. Akimkin |
author_facet |
Marina A. Tyumentseva Aleksandr I. Tyumentsev Vasiliy G. Akimkin |
author_sort |
Marina A. Tyumentseva |
title |
Protocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells |
title_short |
Protocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells |
title_full |
Protocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells |
title_fullStr |
Protocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells |
title_full_unstemmed |
Protocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells |
title_sort |
protocol for assessment of the efficiency of crispr/cas rnp delivery to different types of target cells |
publisher |
Public Library of Science (PLoS) |
publishDate |
2021 |
url |
https://doaj.org/article/29be6d3838f34a0492e637a62af0d06e |
work_keys_str_mv |
AT marinaatyumentseva protocolforassessmentoftheefficiencyofcrisprcasrnpdeliverytodifferenttypesoftargetcells AT aleksandrityumentsev protocolforassessmentoftheefficiencyofcrisprcasrnpdeliverytodifferenttypesoftargetcells AT vasiliygakimkin protocolforassessmentoftheefficiencyofcrisprcasrnpdeliverytodifferenttypesoftargetcells |
_version_ |
1718424467782238208 |