Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK

Abstract CRISPR/Cas9-mediated homology-directed repair (HDR) is used for error-free targeted knock-in of foreign donor DNA. However, the low efficiency of HDR-mediated knock-in hinders establishment of knock-in clones. Double-strand breaks (DSBs) induced by CRISPR/Cas9 are preferentially repaired by...

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Auteurs principaux:	Daisuke Arai, Yoichi Nakao
Format:	article
Langue:	EN
Publié:	Nature Portfolio 2021
Sujets:	Medicine R Science Q
Accès en ligne:	https://doaj.org/article/521d79376b974da7b871454edeb53d3c
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spelling	oai:doaj.org-article:521d79376b974da7b871454edeb53d3c2021-12-02T18:50:48ZEfficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK10.1038/s41598-021-97579-82045-2322https://doaj.org/article/521d79376b974da7b871454edeb53d3c2021-09-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-97579-8https://doaj.org/toc/2045-2322Abstract CRISPR/Cas9-mediated homology-directed repair (HDR) is used for error-free targeted knock-in of foreign donor DNA. However, the low efficiency of HDR-mediated knock-in hinders establishment of knock-in clones. Double-strand breaks (DSBs) induced by CRISPR/Cas9 are preferentially repaired by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) before HDR can occur, thereby preventing HDR-mediated knock-in. NHEJ/MMEJ also cause random integrations, which give rise to false-positive knock-in events, or silently disrupt the genome. In this study, we optimized an HDR-mediated knock-in method for mouse embryonic stem cells (mESCs). We succeeded in improving efficiency of HDR-mediated knock-in of a plasmid donor while almost completely suppressing NHEJ/MMEJ-based integration by combining in vivo-linearization of the donor plasmid, transient knockdown of DNA polymerase θ, and chemical inhibition of DNA-dependent protein kinase (DNA-PK) by M3814. This method also dramatically improved the efficiency of biallelic knock-in; at the Rosa26a locus, 95% of HDR-mediated knock-in clones were biallelic. We designate this method BiPoD (Biallelic knock-in assisted by Pol θ and DNA-PK inhibition). BiPoD achieved simultaneous efficient biallelic knock-in into two loci. BiPoD, therefore, enables rapid and easy establishment of biallelic knock-in mESC lines.Daisuke AraiYoichi NakaoNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-15 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Daisuke Arai Yoichi Nakao Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK
description	Abstract CRISPR/Cas9-mediated homology-directed repair (HDR) is used for error-free targeted knock-in of foreign donor DNA. However, the low efficiency of HDR-mediated knock-in hinders establishment of knock-in clones. Double-strand breaks (DSBs) induced by CRISPR/Cas9 are preferentially repaired by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) before HDR can occur, thereby preventing HDR-mediated knock-in. NHEJ/MMEJ also cause random integrations, which give rise to false-positive knock-in events, or silently disrupt the genome. In this study, we optimized an HDR-mediated knock-in method for mouse embryonic stem cells (mESCs). We succeeded in improving efficiency of HDR-mediated knock-in of a plasmid donor while almost completely suppressing NHEJ/MMEJ-based integration by combining in vivo-linearization of the donor plasmid, transient knockdown of DNA polymerase θ, and chemical inhibition of DNA-dependent protein kinase (DNA-PK) by M3814. This method also dramatically improved the efficiency of biallelic knock-in; at the Rosa26a locus, 95% of HDR-mediated knock-in clones were biallelic. We designate this method BiPoD (Biallelic knock-in assisted by Pol θ and DNA-PK inhibition). BiPoD achieved simultaneous efficient biallelic knock-in into two loci. BiPoD, therefore, enables rapid and easy establishment of biallelic knock-in mESC lines.
format	article
author	Daisuke Arai Yoichi Nakao
author_facet	Daisuke Arai Yoichi Nakao
author_sort	Daisuke Arai
title	Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK
title_short	Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK
title_full	Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK
title_fullStr	Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK
title_full_unstemmed	Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK
title_sort	efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of dna polymerase θ/dna-pk
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/521d79376b974da7b871454edeb53d3c
work_keys_str_mv	AT daisukearai efficientbiallelicknockininmouseembryonicstemcellsbyinvivolinearizationofdonorandtransientinhibitionofdnapolymerasethdnapk AT yoichinakao efficientbiallelicknockininmouseembryonicstemcellsbyinvivolinearizationofdonorandtransientinhibitionofdnapolymerasethdnapk
_version_	1718377502584340480

Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK

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