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 protoc...

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Autores principales:	Marina A Tyumentseva, Aleksandr I Tyumentsev, Vasiliy G Akimkin
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/70b9bfca90db42ca94f533254e8514a5
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spelling	oai:doaj.org-article:70b9bfca90db42ca94f533254e8514a52021-12-02T20:07:42ZProtocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells.1932-620310.1371/journal.pone.0259812https://doaj.org/article/70b9bfca90db42ca94f533254e8514a52021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0259812https://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, p e0259812 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	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/70b9bfca90db42ca94f533254e8514a5
work_keys_str_mv	AT marinaatyumentseva protocolforassessmentoftheefficiencyofcrisprcasrnpdeliverytodifferenttypesoftargetcells AT aleksandrityumentsev protocolforassessmentoftheefficiencyofcrisprcasrnpdeliverytodifferenttypesoftargetcells AT vasiliygakimkin protocolforassessmentoftheefficiencyofcrisprcasrnpdeliverytodifferenttypesoftargetcells
_version_	1718375247992848384

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

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