GMO molecular characterization details

GMO detailed information

GMO:
GE-Low pungency mustard
Alias:

GE mustard, gene editing
UID:
Tradename:
Companies/Institutions:
Pairwise
Species:
Brassica juncea
(brown mustard, Chinese mustard, oriental mustard, Indian mustard)
Traits:
EU authorisation:

no Methods could be found.
no Reference materials could be found
No results found.
no Literature reference could be found

General Information

Description:

GE-Low pungency mustard was developed by editing 17 functional copies of the type-I myrosinase multigene family using CRISPR/Cas12a in Brassica juncea cv. Red Giant delivered through Agrobacterium-mediated transformation.

The intended phenotype of GE-Low pungency mustard is an improved flavour of leafy greens by a reduction of pungency. Rupturing cells of mustard greens lead to contact of normally physically separated molecules – myrosinase enzymes and glucosinolate precursors - leading to hydrolysis of the latter and the production of bitter compounds. To inhibit this ‘mustard bomb’ reaction, multiple myrosinase genes were edited in GE-Low pungency mustard resulting in loss-of-function alleles (see Inserts / Modification for details).

The binary plasmid, pWISE687 was used to express the Lachnospiraceae bacterium Cas12a endonuclease (LbCas12a) and 7 guide RNAs (gRNA), which were designed to target exon VI in multiple myrosinase genes in both the A and B genome.

The developer of this event requested confirmation with USDA APHIS that a Low pungency mustard event is not a regulated article subject to APHIS oversight under 7. C.F.R. part 340. Due to confidential business information in the inquiry letter, it is unclear whether this event resembles the GE-Low pungency Mustard as published by the same developer in a peer-reviewed article (Karlson et al., 2022).

All transgenic elements were removed by segregation and the final event was screened to be transgene-free via target-capture sequencing.

Sources:

USDA-APHIS letter of inquiry 20-168-07_air
Karlson et al. (2022)Targeted Mutagenesis of the Multicopy Myrosinase Gene Family in Allotetraploid Brassica juncea Reduces Pungency in Fresh Leaves across Environments. Plants (Basel) 11(19):2494.

Links regarding to this GMO:

USDA APHIS https://www.aphis.usda.gov/biotechnology/downloads/reg_loi/20-168-07_air_inquiry_a3_a2_cbidel.pdf ,

Scientific publication - Karlsron et al. (2022) https://www.mdpi.com/2223-7747/11/19/2494

Transformation / Modification technique:

CRISPR/Cas; Agrobacterium-induced

Inserts / Modification

Name:	edited_Low pungency mustard
Comment:	As described in Karlson et al. (2022) a total of 17 functional and expressed myrosinase genes were identified. These genes are distributed in both the A and B genomes across seven chromosomes (A01, A02, A03, A09, B04, B05, B07). All of the 17 genes were edited. Seven genes contained small deletions (≤25 bp), five contained large deletions (up to 3781 bp), and 5 were part of a large structural variation (37 kbp deletion, 100kbp inversion).
Map:

Genetic Elements:

Reference: scientific journal

Elements:

Order	Name	Donor	Position	Definition	Trait	Comment
1	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene A01.1_rg; 7 bp deletion leading to a pre-mature stop codon
2	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene A02.1_rg; 365 bp deletion leading to a pre-mature stop codon ans loss of at least 2 active site residues
3	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene A02.2_rg; 1923 bp deletion leading to loss of multiple exons
4	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene A02.3_rg; a 10 bp and a 3971 bp deletion leading to loss of multiple exons
5	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene A02.4_rg; a 93 bp and 8 bp deletion leading to a pre-mature stop codon
6	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene A03.1_rg; two 7 bp deletions leading to a pre-mature stop codon
7	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene A09.1_rg; an 8 bp, a 13 bp, and a 4 bp deletion leading to a pre-mature stop codon
8	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene A09_cl2.1_rg; an 8 bp deletion leading to a pre-mature stop codon
9	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene A09_cl2.2_rg; an 8 bp and a 6 bp deletion leading to a pre-mature stop codon
10	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene B04_cl1.1_rg; whole gene deletion
11	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene B04_cl1.2_rg; whole gene deletion
12	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene B04_cl1.3_rg; B04 gene chimera with a premature stop codon resulting from inversion of a 100 kbp and deletion of a 37 kbp genomic fragment
13	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene B04_cl1.4_rg; B04 gene chimera with a premature stop codon resulting from inversion of a 100 kbp and deletion of a 37 kbp genomic fragment
14	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene B04_cl1.5_rg; B04 gene chimera with a premature stop codon resulting from inversion of a 100 kbp and deletion of a 37 kbp genomic fragment
15	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene B04_cl2.1_rg; 316 bp deletion leading to loss of 1 active site residue
16	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene B07.1_rg; an 8 bp and a 5 bp deletion leading to a pre-mature stop codon
17	CS-myr_genome edited-BRAJU	Brassica juncea	-	Gene from the type-I myrosinase multigene family. Karlson et al. (2022). Plants, 11(19):2494. doi: 10.3390/plants11192494.	Altered flavour	endogenous gene B05; 21 bp deletion leading to loss of one active site residue

Related DNA Sequences:

Acc.No. Reference Sequence Size (bp) Annotation

scientific journal No

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