GMO molecular characterization details

GMO detailed information

GMO:
DP915635
Alias:

915635, GE maize, gene editing
UID:
DP-915635-4
Tradename:
Companies/Institutions:
Pioneer Hi-Bred International, Corteva Agriscience
Species:
Zea mays
(maize, corn)
Traits:
EU authorisation:

General Information

Description:

Maize event DP915635 was produced in a two-step transformation process using biolistic and Agrobacterium-mediated transformation with the vectors PHP73878 and PHP83175.

DP915635 maize is tolerant to the herbicide glufosinate and produces an insecticidal toxin found in specific ferns growing on trees.

In the first step, a site-specific insertion landing pad (SSILP) was introduced into the maize genome using CRISPR/Cas9-mediated homology-directed repair (Gao et al., 2020). In the second step, the integrated landing pad DNA was replaced by the T-DNA via flippase recognition target sites (FRTs).

The T-DNA contained three cassettes:

the ipd079Ea cassette (CS-ipd079Ea_OPHPE) that confers resistance to corn rootworm pests,
the pat cassette (CS-pat-STRVR) that confers tolerance to glufosinate herbicide,
the pmi cassette (CS-pmi-ECOLX) that was used as a selectable marker during transformation.

Southern-by-Sequencing demonstrated that the final event contains a single copy of the inserted DNA derived from PHP83175 and PHP73878, and that no additional insertions or plasmid backbone sequences are present in its genome. Genetic and phenotypic stability of the inserts was confirmed by molecular and segregation analysis of different DP915635 generations.

Sources:

EFSA document - Application submitted under regulation (EC) No 1829/2003: EFSA-Q-2020-00834 on Part VII - Summary of EFSA-GMO-NL-2020-172
USDA-APHIS regulatory status review 24-079-01rsr

Links regarding to this GMO:

Scientific publication - Gao et al. (2020) https://www.frontiersin.org/articles/10.3389/fpls.2020.00535/full,

EFSA https://open.efsa.europa.eu/study-inventory/EFSA-Q-2020-00834,

BCH https://bch.cbd.int/en/database/record?documentID=260914,

USDA-APHIS https://www.aphis.usda.gov/sites/default/files/24-079-01rsr.pdf

Transformation / Modification technique:

CRISPR/Cas; Agrobacterium-induced

Inserts / Modification

Name:	insert_DP915635
Comment:	The vectors PHP73878 and PHP83175 have been used for the creation of event DP915635. The inserted landing pad sequence from PHP73878 was partially replaced with three gene cassettes from the plasmid PHP83175 T-DNA region via recombination.
Map:

Genetic Elements:

Reference: USDA-APHIS

Elements:

Order	Name	Donor	Size (bp)	Position	Definition	Trait	Comment
1	V-loxP-BPP1	Bacteriophage P1	34	135-168
2	P-ubi1-MAIZE	Zea mays	983	181-1163	Promoter region of the polyubiquitin gene. Christensen et al. (1992). Plant Mol Biol, 18:675-689.		including 5' untranslated region
3	I-1_ubi1-MAIZE	Zea mays	1013	1164-2176
4	vector fragment		48	2199-2246			Flippase recombination target site from Saccharomyces cerevisiae (Proteau et al., 1986; GenBank accession NC_001398.1)
5	CS-pmi-ECOLX	Escherichia coli	1216	2265-3480	Phosphomannose isomerase. Reed et al (2001). In Vitro Cell Dev Biol Plant, 37:127-132.	Mannose metabolism	including 5' untranslated region (4 bp) and 3' untranslated region (6 bp)
6	T-pinII-SOLTU	Solanum tuberosum	311	3491-3801	3' untranslated region (3' UTR) of the proteinase inhibitor II gene; 0.31kb terminator sequence.
7	T-z19-MAIZE	Zea mays	742	3812-4553	Terminator of the 19-kDA zein gene from Zea mays. GenBank KX247647.
8	P-act2-ORYSA	Oryza sativa	1682	4757-6438	Promoter of the actin 2 gene from Oryza sativa. GenBank EU155408.1.
9	I-act2-ORYSA	Oryza sativa	469	6439-6907	Intron of the actin-2 gene from Oryza sativa. GenBank EU155408.1
10	CS-pat-STRVR	Streptomyces viridochromogenes	552	6923-7474	Phosphinothricin acetyltransferase gene derived from Streptomyces viridochromogenes a.k.a. pat gene; shares 85 per cent homology at the amino acid level with the bar gene. Wohlleben et al. (1988) Gene 70:25-37.	Glufosinate tolerance	Maize-optimized.
11	T-35S-CaMV	Cauliflower mosaic virus	194	7493-7686	3' untranslated region (3' UTR) of the 35S gene from Cauliflower mosaic virus. Gardner et al. (1981). Nucleic Acids Res, 9:2871-2888.
12	V-loxP-BPP1	Bacteriophage P1	34	7708-7741
13	T-ubi-SORBI	Sorghum bicolor	584	7838-8421	Terminator of the ubiquitin gene. Phytozome gene ID Sobic.004G049900.l.		US Patent 9725731 (Abbitt, 2017)
14	T-gkaf-SORBI	Sorghum bicolor	464	8463-8926	Terminator of the gamma-kafirin gene; De Freitas et al. (1994). Molecular and General Genetics, 245:177-186.		GenBank accession X62480.1
15	E-rcc3-SORBI	Sorghum bicolor	1581	9022-10602	Enhancer region from Sorghum bicolor root cortical RCc3 gene. Patent WO2012112411A1 Diehn and Peterson-Burch, 2012; GenBank JB072168.1.
16	E-rcc3-SORBI	Sorghum bicolor	1581	10609-12189	Enhancer region from Sorghum bicolor root cortical RCc3 gene. Patent WO2012112411A1 Diehn and Peterson-Burch, 2012; GenBank JB072168.1.
17	E-rcc3-SORBI	Sorghum bicolor	1584	12203-13786	Enhancer region from Sorghum bicolor root cortical RCc3 gene. Patent WO2012112411A1 Diehn and Peterson-Burch, 2012; GenBank JB072168.1.
18	P-pcoa-MAIZE	Zea mays	911	13787-14697	Promoter region upstream of a Zea mays PCO118362 mRNA sequence.		Seq ID No: 4 in WO Patent 2017/222821(Crow et al., 2017)
19	I-unknown protein-MAIZE	Zea mays	856	14716-15571	Intron region from the Zea mays ortholog of an Oryza sativa hypothetical protein.		predicted Zea mays (maize) calmodulin 5 gene (Phytozome gene ID Zm00008a029682, WO Patent 2016109157 (Abbitt and Shen, 2016)); Genbank accession LQ422921.1)
20	CS-ipd079Ea_OPHPE	Ophioderma pendulum	1440	15581-17020	Encodes insecticidal protein ipd079Ea from the fern Ophioglossum pendulum. Patent WO 2017023486A1. GenBank ON872186.1	Coleoptera resistance	Seq ID No. 55 in WO Patent 2017/023486 (Allen et al., 2017)
21	T-sci_1b-SORBI	Sorghum bicolor	953	17038-17990	Terminator region of the subtilisin-chymotrypsin inhibitor 1B gene; Patent WO2018102131; GenBank LQ750838.1.
22	T-gz27-MAIZE	Zea mays	460	18037-18496	Terminator of the 27-kDA gamma zein gene.		GenBank accession GX289463.1
23	T-ubi14-ARATH	Arabidopsis thaliana	902	18503-19404	3' untranslated region (3' UTR) of the ubiquitin 14 gene. Callis et al. (1995). Genetics 139:921-939.		GenBank accession NM_001125450.2
24	T-In2_1-MAIZE	Zea mays	494	19411-19904	Terminator region from the Zea mays In2-1 gene. Hershey and Stoner (1991), Plant Mol Biol 17(4):679-90. doi: 10.1007/BF00037053.		GenBank accession AR014639
25	vector fragment		24	19962-19985			attB2: Bacteriophage lambda integrase recombination site from the Invitrogen Gateway® cloning system (Hartley et al., 2000; Katzen, 2007)
26	vector fragment		21	20133-20153			attB3: Bacteriophage lambda integrase recombination site (Cheo et al., 2004)
27	vector fragment		48	20389-20436			FRT6: Modified flippase recombination target site from Saccharomyces cerevisiae (Proteau et al., 1986; GenBank accession NC_001398.1)

Related DNA Sequences:

Acc.No. Reference Sequence Size (bp) Annotation

USDA-APHIS Yes 20564 Sequence of the inserted genetic material - DP915635

Acc.No.	Reference	Sequence	Size (bp)	Annotation
	USDA-APHIS	Yes	20564	Sequence of the inserted genetic material - DP915635

Detection Methods

17 results

Name	Type	Target	Description	Validation	Standardisation	Verification
QL-ELE-AINT 2-5'/AINT 2-3'	element-specific	I-actin	Qualitative real-time PCR (TaqMan) method for detection of rice actin 1 intron (I-ractI) (Mano et al. 2009).	in-house validation	none	1
QL-ELE-P-Ubi-F/P-Ubi-R	element-specific	P-ubi1-MAIZE	Qualitative real-time PCR (TaqMan) method for detection of ubiquitin promoter of Zea mays (Debode et al., 2013)	in-house validation	none	1
QL-ELE-Patf-n2/Patr-n2	element-specific	CS-pat-STRVR	Qualitative real-time PCR (TaqMan) method for detection of the pat gene (Xu et al., 2006)	in-house validation	none	1
QL-ELE-T-35S-F/T-35S-R	element-specific	T-Cauliflower mosaic virus	Qualitative real-time PCR (TaqMan) method for detection of the 35S terminator from Cauliflower mosaic virus (Debode et al., 2013)	in-house validation	none	1
QL-TAX-ZM-002	taxon-specific	Zea mays	Qualitative real-time PCR (SYBRGreen) method for detection of maize alcoholdeydrogenase 1 gene (Barbau-Piednoir et al., 2014)	ring trial validation	unknown	1
QL-TAX-ZM-003	taxon-specific	Zea mays	Qualitative conventional PCR method for detection of the maize invertase gene (ISO 21569)	ring trial validation	ISO standard	1
QT-EVE-ZM-031	event-specific	DP915635	Quantitative PCR method for detection of maize event DP915635 (EU-RL GMFF GMOMETHODS database as of May 5, 2023).	ring trial validation	EU reference method	3
QT-TAX-ZM-001	taxon-specific	Zea mays	Quantitative real-time PCR (TaqMan) method for detection of maize alcohol dehydrogenase 1 (adh1) gene. Estimated number of target copies per haploid genome (Jacchia et al. 2018): 1	ring trial validation	ISO standard	1
QT-TAX-ZM-002	taxon-specific	Zea mays	Quantitative real-time PCR (TaqMan) method for detection of the maize high-mobility-group A (hmgA) gene (ISO 21570). Estimated number of target copies per haploid genome (Jacchia et al. 2018): 1	ring trial validation	ISO standard	1
QT-TAX-ZM-003	taxon-specific	Zea mays	Quantitative real-time PCR (TaqMan) method for detection of maize alcohol dehydrogenase 1 (adh1) gene (EU-RL GMFF GMOMETHODS database as of October 01, 2014)	ring trial validation	EU reference method	1
QT-TAX-ZM-004	taxon-specific	Zea mays	Quantitative real-time PCR (TaqMan) for detection of maize aldolase 1 (ald1) gene (EU-RL GMFF GMOMETHODS database as of July 14, 2016). Estimated number of target copies per haploid genome (Jacchia et al. 2018): 2	ring trial validation	EU reference method	1
QT-TAX-ZM-005	taxon-specific	Zea mays	Quantitative PCR method for detection of maize high-mobility-group (hmg) gene (EU-RL GMFF GMOMETHODS database as of May 28, 2018).	ring trial validation	EU reference method	1
QT-TAX-ZM-006	taxon-specific	Zea mays	Quantitative real-time PCR (TaqMan) method for detection of maize starch synthase IIb gene (ISO 21570)	ring trial validation	ISO standard	1
QT-TAX-ZM-008	taxon-specific	Zea mays	Quantitative real-time PCR method for detection of maize high-mobility-group (hmg) gene (EU-RL GMFF GMOMETHODS database as of July 20, 2023). Estimated number of target copies per haploid genome (Jacchia et al. 2018): 1	ring trial validation	EU reference method	1
QT-TAX-ZM-011	taxon-specific	Zea mays	Quantitative real-time PCR (TaqMan) method for detection of the maize alcohol dehydrogenase 1 gene (EU-RL GMFF GMOMETHODS database as of March 06, 2014)	ring trial validation	EU reference method	1
QT-TAX-ZM-014	taxon-specific	Zea mays	Quantitative real-time PCR (TaqMan) method for detection of maize alcohol dehydrogenase 1 gene (EU-RL GMFF GMOMETHODS database as of March 06, 2014). Estimated number of target copies per haploid genome (Jacchia et al. 2018): 1	ring trial validation	EU reference method	1
QT-TAX-zSSIIb-3F/zSSIIb-4R	taxon-specific	Zea mays	Quantitative real-time PCR (TaqMan) method for detection of maize starch synthase IIb gene (Yang et al., 2005)	unknown	unknown	1

Catalogue number	Source	Value	Type	Certified	Available
ERM-BF447a	JRC - CRM (ERM)	0.0 % (m/m)	dried powder	yes	yes
ERM-BF447b	JRC - CRM (ERM)	99 % (m/m)	dried powder	yes	yes
ERM-BF447c	JRC - CRM (ERM)	0.099 % (m/m)	dried powder	yes	yes
ERM-BF447d	JRC - CRM (ERM)	0.99 % (m/m)	dried powder	yes	yes
ERM-BF447e	JRC - CRM (ERM)	9.9 % (m/m)	dried powder	yes	yes

Citation	Publication year	Publishing authority	Publication type	Content types
Assessment of genetically modified maize DP915635 for food and feed uses, under Regulation (EC) No 1829/2003 (application EFSA‐GMO‐NL‐2020‐172)	2024	EFSA GMO Panel	Scientific opinion EFSA GMO Panel	risk assessment document
Commission Implementing Decision (EU) 2024/1822 of 2 July 2024 authorising the placing on the market of products containing, consisting of or produced from genetically modified maize DP915635 pursuant to Regulation (EC) No 1829/2003 of the European Parliament and of the Council	2024	EU	EU Commission Decision	decision document
Application for authorisation of genetically modified plants and derived food and feed in accordance with regulation (EC) No 1829/2003, DP915635 maize, EFSA-GMO-NL-2020-172, Part VII - Summary	2020	EFSA	application summary	application summary

European GMO Initiative for a Unified Database System

GMO detailed information

GMO:

Alias:

UID:

Tradename:

Companies/Institutions:

Species:

Traits:

EU authorisation:

General Information

Links regarding to this GMO:

Inserts / Modification

Transformation Vectors

Detection Methods

Reference Materials

Authorisation

Literature reference