CRISPR/Cas9-Mediated Knockouts of the ALG3 and GNTI in N. benthamiana and Their Application to Pharmaceutical Production

Bataa, Dolgormaa; Kajiura, Hiroyuki; Sawada‐Choi, Reimi Lai Sang; Yamashita, Yukino; Ishimizu, Takeshi; Misaki, Ryo; Takeda, Atsushi; Fujiyama, Kazuhito

doi:https://doi.org/10.1111/pbi.70326

CRISPR/Cas9-Mediated Knockouts of the ALG3 and GNTI in N. benthamiana and Their Application to Pharmaceutical Production

Bataa, Dolgormaa; Kajiura, Hiroyuki; Sawada‐Choi, Reimi Lai Sang et al.

Plant Biotechnology Journal, 2025, 6, 439

Number of Access:21（2025-10-24 15:20 Counts）

Identifier to cite or link to this item: https://hdl.handle.net/11094/102750

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Title	CRISPR/Cas9-Mediated Knockouts of the ALG3 and GNTI in N. benthamiana and Their Application to Pharmaceutical Production
Creator	CreatorName Bataa, Dolgormaa
	NRID 1000030704209 1000030704209 ORCID 0000-0001-5811-1625 0000-0001-5811-1625 researchmap permalink 7000020437 7000020437 Scopus Author ID 16241653300 16241653300 CreatorName Kajiura, Hiroyuki
	CreatorName Sawada‐Choi, Reimi Lai Sang
	CreatorName Yamashita, Yukino
	NRID 1000030314355 1000030314355 CreatorName Ishimizu, Takeshi
	NRID 1000020571186 1000020571186 researchmap permalink read0090369 read0090369 Scopus Author ID 6603301567 6603301567 CreatorName Misaki, Ryo
	NRID 1000060560779 1000060560779 CreatorName Takeda, Atsushi
	NRID 1000070209112 1000070209112 researchmap permalink read0014155 read0014155 Scopus Author ID 7005164172 7005164172 CreatorName Fujiyama, Kazuhito
Subject	α1,3-mannosyltransferase
	CRISPR/Cas9
	N-acetylglucosaminyltransferase I
	N-glycoengineering
	Nicotiana benthamiana
Description	Bataa Dolgormaa, Kajiura Hiroyuki, Sawada‐Choi Reimi Lai Sang, et al. CRISPR/Cas9-Mediated Knockouts of the ALG3 and GNTI in N. benthamiana and Their Application to Pharmaceutical Production. Plant Biotechnology Journal 6, 439 (2025); https://doi.org/10.1111/pbi.70326.
Abstract	N-Glycosylation critically influences the efficacy, safety and pharmacokinetic properties of biopharmaceuticals. Plant expression platforms offer multiple advantages for the production of N-glycosylated proteins, but their use is impeded by the presence of plant-specific N-glycan epitopes, which raise concerns of possible immunogenicity to humans. In this study, N-glycoengineered Nicotiana benthamiana plants that produce more homogeneous N-glycans without plant-specific epitopes were generated using multiplex CRISPR/Cas9 genome editing. To achieve this N-glycosylation modification, ALG3 and GNTI, which function in N-glycosylation processes in the ER and Golgi, respectively, were characterised, and single- and double-knockout mutant plants were generated. Comprehensive N-glycan profiling revealed that while the ALG3-knockout plant line, alg3, maintained predominantly plant-specific N-glycans with altered mannose content, the GNTI-knockout line, gntI, produced exclusively high-mannose-type N-glycans. Notably, the alg3gntI double-knockout mutants yielded highly uniform trimannosidic N-glycans. To validate our N-glycoengineering approach, we expressed two model biopharmaceuticals, Varlilumab (anti-CD27 antibody) and β-glucocerebrosidase (GCase), in wild-type and mutant plants. While the antibodies expressed in alg3 and alg3gntI showed a certain level of glucosylated endoplasmic reticulum-type N-glycan, with increased non-N-glycosylated heavy chains, GCase exhibited a more consistent N-glycosylation profile, reflecting the engineered N-glycosylation pathway. Our findings provide valuable insights into N-glycan biosynthesis in N. benthamiana and demonstrate the potential of targeted N-glycoengineering for producing biopharmaceuticals with more homogeneous mannose-type N-glycan profiles.
Publisher	Wiley
Source Title	Plant Biotechnology Journal
Volume (Issue)	6
Page	439
Date of Issued	2025-09-04
Language	English
Handle URL	https://hdl.handle.net/11094/102750
PISSN	14677644
eISSN	14677652
Relation.isIdenticalTo	DOI (Publisher Version) https://doi.org/10.1111/pbi.70326
Access Rights	open access
CopyRight	This article is licensed under a Creative Commons Attribution 4.0 International License.
oaire:version	Version of Record
Category	学術雑誌論文 Journal Article

Resource Type	journal article
Local Type	Journal Article
DCMI Type	Text
DCTERMS.bibliographicCitation	Plant Biotechnology Journal. 2025, 6, p. 439
DC.title	CRISPR/Cas9-Mediated Knockouts of the ALG3 and GNTI in N. benthamiana and Their Application to Pharmaceutical Production
DC.creator	Bataa, Dolgormaa
	Kajiura, Hiroyuki
	Sawada‐Choi, Reimi Lai Sang
	Yamashita, Yukino
	Ishimizu, Takeshi
	Misaki, Ryo
	Takeda, Atsushi
	Fujiyama, Kazuhito
DC.publisher	Wiley
DC.language' scheme='DCTERMS.RFC1766	English
DC.type' scheme='DCTERMS.DCMIType	Text
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2025-09-04
DC.identifier	https://doi.org/10.1111/pbi.70326
DC.subject	α1,3-mannosyltransferase
	CRISPR/Cas9
	N-acetylglucosaminyltransferase I
	N-glycoengineering
	Nicotiana benthamiana
DC.description	Bataa Dolgormaa, Kajiura Hiroyuki, Sawada‐Choi Reimi Lai Sang, et al. CRISPR/Cas9-Mediated Knockouts of the ALG3 and GNTI in N. benthamiana and Their Application to Pharmaceutical Production. Plant Biotechnology Journal 6, 439 (2025); https://doi.org/10.1111/pbi.70326.
DCTERMS.abstract	N-Glycosylation critically influences the efficacy, safety and pharmacokinetic properties of biopharmaceuticals. Plant expression platforms offer multiple advantages for the production of N-glycosylated proteins, but their use is impeded by the presence of plant-specific N-glycan epitopes, which raise concerns of possible immunogenicity to humans. In this study, N-glycoengineered Nicotiana benthamiana plants that produce more homogeneous N-glycans without plant-specific epitopes were generated using multiplex CRISPR/Cas9 genome editing. To achieve this N-glycosylation modification, ALG3 and GNTI, which function in N-glycosylation processes in the ER and Golgi, respectively, were characterised, and single- and double-knockout mutant plants were generated. Comprehensive N-glycan profiling revealed that while the ALG3-knockout plant line, alg3, maintained predominantly plant-specific N-glycans with altered mannose content, the GNTI-knockout line, gntI, produced exclusively high-mannose-type N-glycans. Notably, the alg3gntI double-knockout mutants yielded highly uniform trimannosidic N-glycans. To validate our N-glycoengineering approach, we expressed two model biopharmaceuticals, Varlilumab (anti-CD27 antibody) and β-glucocerebrosidase (GCase), in wild-type and mutant plants. While the antibodies expressed in alg3 and alg3gntI showed a certain level of glucosylated endoplasmic reticulum-type N-glycan, with increased non-N-glycosylated heavy chains, GCase exhibited a more consistent N-glycosylation profile, reflecting the engineered N-glycosylation pathway. Our findings provide valuable insights into N-glycan biosynthesis in N. benthamiana and demonstrate the potential of targeted N-glycoengineering for producing biopharmaceuticals with more homogeneous mannose-type N-glycan profiles.
DC.rights	This article is licensed under a Creative Commons Attribution 4.0 International License.
DC.format	102750
citation_title	CRISPR/Cas9-Mediated Knockouts of the ALG3 and GNTI in N. benthamiana and Their Application to Pharmaceutical Production
citation_author	Bataa, Dolgormaa
	Kajiura, Hiroyuki
	Sawada‐Choi, Reimi Lai Sang
	Yamashita, Yukino
	Ishimizu, Takeshi
	Misaki, Ryo
	Takeda, Atsushi
	Fujiyama, Kazuhito
citation_publisher	Wiley
citation_language	English
citation_date	2025-09-04
citation_journal_title	Plant Biotechnology Journal
citation_volume	6
citation_firstpage	439
citation_doi	https://doi.org/10.1111/pbi.70326
citation_public_url	https://hdl.handle.net/11094/102750

CRISPR/Cas9-Mediated Knockouts of the ALG3 and GNTI in N. benthamiana and Their Application to Pharmaceutical Production

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