‘Cold shock’ increases the frequency of homology directed repair gene editing in induced pluripotent stem cells

Abstract Using CRISPR/Cas9 delivered as a RNA modality in conjunction with a lipid specifically formulated for large RNA molecules, we demonstrate that homology directed repair (HDR) rates between 20–40% can be achieved in induced pluripotent stem cells (iPSC). Furthermore, low HDR rates (between 1–...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Q. Guo, G. Mintier, M. Ma-Edmonds, D. Storton, X. Wang, X. Xiao, B Kienzle, D. Zhao, John N. Feder
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/7f2a87b2a216424889cfd9d640ef048d
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:7f2a87b2a216424889cfd9d640ef048d
record_format	dspace
spelling	oai:doaj.org-article:7f2a87b2a216424889cfd9d640ef048d2021-12-02T15:08:03Z‘Cold shock’ increases the frequency of homology directed repair gene editing in induced pluripotent stem cells10.1038/s41598-018-20358-52045-2322https://doaj.org/article/7f2a87b2a216424889cfd9d640ef048d2018-02-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-20358-5https://doaj.org/toc/2045-2322Abstract Using CRISPR/Cas9 delivered as a RNA modality in conjunction with a lipid specifically formulated for large RNA molecules, we demonstrate that homology directed repair (HDR) rates between 20–40% can be achieved in induced pluripotent stem cells (iPSC). Furthermore, low HDR rates (between 1–20%) can be enhanced two- to ten-fold in both iPSCs and HEK293 cells by ‘cold shocking’ cells at 32 °C for 24–48 hours following transfection. This method can also increases the proportion of loci that have undergone complete sequence conversion across the donor sequence, or ‘perfect HDR’, as opposed to partial sequence conversion where nucleotides more distal to the CRISPR cut site are less efficiently incorporated (‘partial HDR’). We demonstrate that the structure of the single-stranded DNA oligo donor can influence the fidelity of HDR, with oligos symmetric with respect to the CRISPR cleavage site and complementary to the target strand being more efficient at directing ‘perfect HDR’ compared to asymmetric non-target strand complementary oligos. Our protocol represents an efficient method for making CRISPR-mediated, specific DNA sequence changes within the genome that will facilitate the rapid generation of genetic models of human disease in iPSCs as well as other genome engineered cell lines.Q. GuoG. MintierM. Ma-EdmondsD. StortonX. WangX. XiaoB KienzleD. ZhaoJohn N. FederNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-11 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Q. Guo G. Mintier M. Ma-Edmonds D. Storton X. Wang X. Xiao B Kienzle D. Zhao John N. Feder ‘Cold shock’ increases the frequency of homology directed repair gene editing in induced pluripotent stem cells
description	Abstract Using CRISPR/Cas9 delivered as a RNA modality in conjunction with a lipid specifically formulated for large RNA molecules, we demonstrate that homology directed repair (HDR) rates between 20–40% can be achieved in induced pluripotent stem cells (iPSC). Furthermore, low HDR rates (between 1–20%) can be enhanced two- to ten-fold in both iPSCs and HEK293 cells by ‘cold shocking’ cells at 32 °C for 24–48 hours following transfection. This method can also increases the proportion of loci that have undergone complete sequence conversion across the donor sequence, or ‘perfect HDR’, as opposed to partial sequence conversion where nucleotides more distal to the CRISPR cut site are less efficiently incorporated (‘partial HDR’). We demonstrate that the structure of the single-stranded DNA oligo donor can influence the fidelity of HDR, with oligos symmetric with respect to the CRISPR cleavage site and complementary to the target strand being more efficient at directing ‘perfect HDR’ compared to asymmetric non-target strand complementary oligos. Our protocol represents an efficient method for making CRISPR-mediated, specific DNA sequence changes within the genome that will facilitate the rapid generation of genetic models of human disease in iPSCs as well as other genome engineered cell lines.
format	article
author	Q. Guo G. Mintier M. Ma-Edmonds D. Storton X. Wang X. Xiao B Kienzle D. Zhao John N. Feder
author_facet	Q. Guo G. Mintier M. Ma-Edmonds D. Storton X. Wang X. Xiao B Kienzle D. Zhao John N. Feder
author_sort	Q. Guo
title	‘Cold shock’ increases the frequency of homology directed repair gene editing in induced pluripotent stem cells
title_short	‘Cold shock’ increases the frequency of homology directed repair gene editing in induced pluripotent stem cells
title_full	‘Cold shock’ increases the frequency of homology directed repair gene editing in induced pluripotent stem cells
title_fullStr	‘Cold shock’ increases the frequency of homology directed repair gene editing in induced pluripotent stem cells
title_full_unstemmed	‘Cold shock’ increases the frequency of homology directed repair gene editing in induced pluripotent stem cells
title_sort	‘cold shock’ increases the frequency of homology directed repair gene editing in induced pluripotent stem cells
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/7f2a87b2a216424889cfd9d640ef048d
work_keys_str_mv	AT qguo coldshockincreasesthefrequencyofhomologydirectedrepairgeneeditingininducedpluripotentstemcells AT gmintier coldshockincreasesthefrequencyofhomologydirectedrepairgeneeditingininducedpluripotentstemcells AT mmaedmonds coldshockincreasesthefrequencyofhomologydirectedrepairgeneeditingininducedpluripotentstemcells AT dstorton coldshockincreasesthefrequencyofhomologydirectedrepairgeneeditingininducedpluripotentstemcells AT xwang coldshockincreasesthefrequencyofhomologydirectedrepairgeneeditingininducedpluripotentstemcells AT xxiao coldshockincreasesthefrequencyofhomologydirectedrepairgeneeditingininducedpluripotentstemcells AT bkienzle coldshockincreasesthefrequencyofhomologydirectedrepairgeneeditingininducedpluripotentstemcells AT dzhao coldshockincreasesthefrequencyofhomologydirectedrepairgeneeditingininducedpluripotentstemcells AT johnnfeder coldshockincreasesthefrequencyofhomologydirectedrepairgeneeditingininducedpluripotentstemcells
_version_	1718388261103075328

‘Cold shock’ increases the frequency of homology directed repair gene editing in induced pluripotent stem cells

Ejemplares similares