Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells

Abstract CRISPR/Cas9 is an efficient customizable nuclease to generate double-strand breaks (DSBs) in the genome. This process results in knockout of the targeted gene or knock-in of a specific DNA fragment at the targeted locus in the genome of various species. However, efficiency of knock-in media...

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Autores principales:	Guoling Li, Xianwei Zhang, Cuili Zhong, Jianxin Mo, Rong Quan, Jie Yang, Dewu Liu, Zicong Li, Huaqiang Yang, Zhenfang Wu
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/1649ef17af0144d78c7d0017adcd1333
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spelling	oai:doaj.org-article:1649ef17af0144d78c7d0017adcd13332021-12-02T12:32:50ZSmall molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells10.1038/s41598-017-09306-x2045-2322https://doaj.org/article/1649ef17af0144d78c7d0017adcd13332017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-09306-xhttps://doaj.org/toc/2045-2322Abstract CRISPR/Cas9 is an efficient customizable nuclease to generate double-strand breaks (DSBs) in the genome. This process results in knockout of the targeted gene or knock-in of a specific DNA fragment at the targeted locus in the genome of various species. However, efficiency of knock-in mediated by homology-directed repair (HDR) pathway is substantially lower compared with the efficiency of knockout mediated by the nonhomologous end-joining (NHEJ) pathway. Suppressing NHEJ pathway or enhancing HDR pathway has been proven to enhance the nuclease-mediated knock-in efficiency in cultured cells and model organisms. We here investigated the effect of small molecules, Scr7, L755507 and resveratrol, on promoting HDR efficiency in porcine fetal fibroblasts. Results from eGFP reporter assay showed that these small molecules could increase the HDR efficiency by 2–3-fold in porcine fetal fibroblasts. When transfecting with the homologous template DNA and CRISPR/Cas9 plasmid and treating with small molecules, the rate of knock-in porcine fetal fibroblast cell lines with large DNA fragment integration could reach more than 50% of the screened cell colonies, compared with 26.1% knock-in cell lines in the DMSO-treated group. The application of small molecules offers a beneficial approach to improve the frequency of precise genetic modifications in primary somatic cells.Guoling LiXianwei ZhangCuili ZhongJianxin MoRong QuanJie YangDewu LiuZicong LiHuaqiang YangZhenfang WuNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Guoling Li Xianwei Zhang Cuili Zhong Jianxin Mo Rong Quan Jie Yang Dewu Liu Zicong Li Huaqiang Yang Zhenfang Wu Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells
description	Abstract CRISPR/Cas9 is an efficient customizable nuclease to generate double-strand breaks (DSBs) in the genome. This process results in knockout of the targeted gene or knock-in of a specific DNA fragment at the targeted locus in the genome of various species. However, efficiency of knock-in mediated by homology-directed repair (HDR) pathway is substantially lower compared with the efficiency of knockout mediated by the nonhomologous end-joining (NHEJ) pathway. Suppressing NHEJ pathway or enhancing HDR pathway has been proven to enhance the nuclease-mediated knock-in efficiency in cultured cells and model organisms. We here investigated the effect of small molecules, Scr7, L755507 and resveratrol, on promoting HDR efficiency in porcine fetal fibroblasts. Results from eGFP reporter assay showed that these small molecules could increase the HDR efficiency by 2–3-fold in porcine fetal fibroblasts. When transfecting with the homologous template DNA and CRISPR/Cas9 plasmid and treating with small molecules, the rate of knock-in porcine fetal fibroblast cell lines with large DNA fragment integration could reach more than 50% of the screened cell colonies, compared with 26.1% knock-in cell lines in the DMSO-treated group. The application of small molecules offers a beneficial approach to improve the frequency of precise genetic modifications in primary somatic cells.
format	article
author	Guoling Li Xianwei Zhang Cuili Zhong Jianxin Mo Rong Quan Jie Yang Dewu Liu Zicong Li Huaqiang Yang Zhenfang Wu
author_facet	Guoling Li Xianwei Zhang Cuili Zhong Jianxin Mo Rong Quan Jie Yang Dewu Liu Zicong Li Huaqiang Yang Zhenfang Wu
author_sort	Guoling Li
title	Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells
title_short	Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells
title_full	Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells
title_fullStr	Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells
title_full_unstemmed	Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells
title_sort	small molecules enhance crispr/cas9-mediated homology-directed genome editing in primary cells
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/1649ef17af0144d78c7d0017adcd1333
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_version_	1718393902302494720

Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells

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