Optimization of genome engineering approaches with the CRISPR/Cas9 system.

Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching...

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Autores principales:	Kai Li, Gang Wang, Troels Andersen, Pingzhu Zhou, William T Pu
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f6caf0c6e1134b9bb7c852fa3574dd9b
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spelling	oai:doaj.org-article:f6caf0c6e1134b9bb7c852fa3574dd9b2021-11-25T06:02:50ZOptimization of genome engineering approaches with the CRISPR/Cas9 system.1932-620310.1371/journal.pone.0105779https://doaj.org/article/f6caf0c6e1134b9bb7c852fa3574dd9b2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/25166277/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching for transfected cells are critical for efficiently recovering modified clones. Paired gRNAs and wild-type Cas9 efficiently create programmed deletions, which facilitate identification of targeted clones, while paired gRNAs and the Cas9D10A nickase generated smaller targeted indels with lower chance of off-target mutagenesis. Genome editing is also useful for programmed introduction of exogenous DNA sequences at a target locus. Increasing the length of the homology arms of the homology-directed repair template strongly enhanced targeting efficiency, while increasing the length of the DNA insert reduced it. Together our data provide guidance on optimal design of scarless gene knockout, modification, or knock-in experiments using Cas9 nuclease.Kai LiGang WangTroels AndersenPingzhu ZhouWilliam T PuPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 8, p e105779 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Kai Li Gang Wang Troels Andersen Pingzhu Zhou William T Pu Optimization of genome engineering approaches with the CRISPR/Cas9 system.
description	Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching for transfected cells are critical for efficiently recovering modified clones. Paired gRNAs and wild-type Cas9 efficiently create programmed deletions, which facilitate identification of targeted clones, while paired gRNAs and the Cas9D10A nickase generated smaller targeted indels with lower chance of off-target mutagenesis. Genome editing is also useful for programmed introduction of exogenous DNA sequences at a target locus. Increasing the length of the homology arms of the homology-directed repair template strongly enhanced targeting efficiency, while increasing the length of the DNA insert reduced it. Together our data provide guidance on optimal design of scarless gene knockout, modification, or knock-in experiments using Cas9 nuclease.
format	article
author	Kai Li Gang Wang Troels Andersen Pingzhu Zhou William T Pu
author_facet	Kai Li Gang Wang Troels Andersen Pingzhu Zhou William T Pu
author_sort	Kai Li
title	Optimization of genome engineering approaches with the CRISPR/Cas9 system.
title_short	Optimization of genome engineering approaches with the CRISPR/Cas9 system.
title_full	Optimization of genome engineering approaches with the CRISPR/Cas9 system.
title_fullStr	Optimization of genome engineering approaches with the CRISPR/Cas9 system.
title_full_unstemmed	Optimization of genome engineering approaches with the CRISPR/Cas9 system.
title_sort	optimization of genome engineering approaches with the crispr/cas9 system.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/f6caf0c6e1134b9bb7c852fa3574dd9b
work_keys_str_mv	AT kaili optimizationofgenomeengineeringapproacheswiththecrisprcas9system AT gangwang optimizationofgenomeengineeringapproacheswiththecrisprcas9system AT troelsandersen optimizationofgenomeengineeringapproacheswiththecrisprcas9system AT pingzhuzhou optimizationofgenomeengineeringapproacheswiththecrisprcas9system AT williamtpu optimizationofgenomeengineeringapproacheswiththecrisprcas9system
_version_	1718414257597448192

Optimization of genome engineering approaches with the CRISPR/Cas9 system.

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