Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells

Tsukamoto, Tomohito; Sakai, Eiko; Nishimae, Fumitaka; Sakurai, Fuminori; Mizuguchi, Hiroyuki

doi:https://doi.org/10.1016/j.jbiotec.2019.08.004

Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells

Tsukamoto, Tomohito; Sakai, Eiko; Nishimae, Fumitaka et al.

Journal of Biotechnology, 2019, 304, 1-9

Number of Access:707（2025-09-01 18:06 Counts）

Identifier to cite or link to this item: https://hdl.handle.net/11094/92344

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Title	Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells
Creator	researchmap permalink tsukamoto-t tsukamoto-t CreatorName Tsukamoto, Tomohito
	CreatorName Sakai, Eiko
	CreatorName Nishimae, Fumitaka
	NRID 1000070370939 1000070370939 researchmap permalink read0165081 read0165081 Scopus Author ID 7003748169 7003748169 CreatorName Sakurai, Fuminori
	NRID 1000050311387 1000050311387 researchmap permalink read0165079 read0165079 Scopus Author ID 57195381326 57195381326 CreatorName Mizuguchi, Hiroyuki
Subject	Adenovirus vector
	Cas12a
	Clustered regularly interspaced short palindromic repeat
Description	Tsukamoto T., Sakai E., Nishimae F., et al. Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells. Journal of Biotechnology 304, 1 (2019); https://doi.org/10.1016/j.jbiotec.2019.08.004.
Abstract	Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas) 9 system is a powerful tool for genome editing and still being aggressively improved. Cas12a, a recently discovered Cas9 ortholog, is expected to become complementary to Cas9 due to its unique characteristics. Previously we attempted to establish an adenovirus (Ad) vector-mediated delivery of CRISPR-Cas12a system since Ad vector is widely used for gene transfer in basic researches and medical applications. However, we found difficulties preparing of Ad vectors at an adequate titer. In this study, we have developed Ad vectors that conditionally express Cas12a either by a tetracycline-controlled promoter or a hepatocyte specific promoter to avoid putative inhibitory effects of Cas12a. These vectors successfully proliferated in packaging cells, HEK293 cells, and were recovered at high titers. We have also developed packaging cells that express shRNA for Cas12a to suppress expression of Cas12a. Using the cells, the Ad vector directing constitutive expression of Cas12a proliferated efficiently and was successfully recovered at a high titer. Overall, we improved recovery of Ad vectors carrying CRISPR-Cas12a system, thus provided them as a tool in genome editing researches.
Publisher	Elsevier B.V.
Source Title	Journal of Biotechnology
Volume (Issue)	304
Page	1-9
Date of Issued	2019-10-10
Language	English
Handle URL	https://hdl.handle.net/11094/92344
PISSN	01681656
NCID	AA11533280
Relation.isVersionOf	DOI (Publisher Version) https://doi.org/10.1016/j.jbiotec.2019.08.004
Access Rights	open access
CopyRight	©2019. This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
oaire:version	Accepted Manuscript
Category	学術雑誌論文 Journal Article

Resource Type	journal article
Local Type	Journal Article
DCMI Type	Text
DCTERMS.bibliographicCitation	Journal of Biotechnology. 2019, 304, p. 1-9
DC.title	Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells
DC.creator	Tsukamoto, Tomohito
	Sakai, Eiko
	Nishimae, Fumitaka
	Sakurai, Fuminori
	Mizuguchi, Hiroyuki
DC.publisher	Elsevier B.V.
DC.language' scheme='DCTERMS.RFC1766	English
DC.type' scheme='DCTERMS.DCMIType	Text
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2019-10-10
DC.identifier	https://doi.org/10.1016/j.jbiotec.2019.08.004
DC.subject	Adenovirus vector
	Cas12a
	Clustered regularly interspaced short palindromic repeat
DC.description	Tsukamoto T., Sakai E., Nishimae F., et al. Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells. Journal of Biotechnology 304, 1 (2019); https://doi.org/10.1016/j.jbiotec.2019.08.004.
DCTERMS.abstract	Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas) 9 system is a powerful tool for genome editing and still being aggressively improved. Cas12a, a recently discovered Cas9 ortholog, is expected to become complementary to Cas9 due to its unique characteristics. Previously we attempted to establish an adenovirus (Ad) vector-mediated delivery of CRISPR-Cas12a system since Ad vector is widely used for gene transfer in basic researches and medical applications. However, we found difficulties preparing of Ad vectors at an adequate titer. In this study, we have developed Ad vectors that conditionally express Cas12a either by a tetracycline-controlled promoter or a hepatocyte specific promoter to avoid putative inhibitory effects of Cas12a. These vectors successfully proliferated in packaging cells, HEK293 cells, and were recovered at high titers. We have also developed packaging cells that express shRNA for Cas12a to suppress expression of Cas12a. Using the cells, the Ad vector directing constitutive expression of Cas12a proliferated efficiently and was successfully recovered at a high titer. Overall, we improved recovery of Ad vectors carrying CRISPR-Cas12a system, thus provided them as a tool in genome editing researches.
DC.rights	©2019. This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
DC.format	92344
citation_title	Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells
citation_author	Tsukamoto, Tomohito
	Sakai, Eiko
	Nishimae, Fumitaka
	Sakurai, Fuminori
	Mizuguchi, Hiroyuki
citation_publisher	Elsevier B.V.
citation_language	English
citation_date	2019-10-10
citation_journal_title	Journal of Biotechnology
citation_volume	304
citation_firstpage	1
citation_lastpage	9
citation_doi	https://doi.org/10.1016/j.jbiotec.2019.08.004
citation_public_url	https://hdl.handle.net/11094/92344

Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells

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