TY - JOUR

T1 - Removing the major allergen Bra j I from brown mustard (Brassica juncea) by CRISPR/Cas9

AU - Assou, Juvenal

AU - Zhang, Dingbo

AU - Roth, Kristian D R

AU - Steinke, Stephan

AU - Hust, Michael

AU - Reinard, Thomas

AU - Winkelmann, Traud

AU - Boch, Jens

N1 - Funding information: This work was supported by funding from the Federal Ministry of Education and Research (BMBF LACOP consortium; grant number 031B0348). The authors thank Ewa Schneider for excellent technical assistance, David Wamhoff for help in statistics, and Jana Streubel for MoClo?compatible genome editing vectors. Open access funding enabled and organized by ProjektDEAL.

PY - 2022/1/29

Y1 - 2022/1/29

N2 - Food allergies are a major health issue worldwide. Modern breeding techniques such as genome editing via CRISPR/Cas9 have the potential to mitigate this by targeting allergens in plants. This study addressed the major allergen Bra j I, a seed storage protein of the 2S albumin class, in the allotetraploid brown mustard (Brassica juncea). Cotyledon explants of an Indian gene bank accession (CR2664) and the German variety Terratop were transformed using Agrobacterium tumefaciens harboring binary vectors with multiple single guide RNAs to induce either large deletions or frameshift mutations in both Bra j I homoeologs. A total of 49 T 0 lines were obtained with up to 3.8% transformation efficiency. Four lines had large deletions of 566 up to 790 bp in the Bra j IB allele. Among 18 Terratop T 0 lines, nine carried indels in the targeted regions. From 16 analyzed CR2664 T 0 lines, 14 held indels and three had all four Bra j I alleles mutated. The majority of the CRISPR/Cas9-induced mutations were heritable to T 1 progenies. In some edited lines, seed formation and viability were reduced and seeds showed a precocious development of the embryo leading to a rupture of the testa already in the siliques. Immunoblotting using newly developed Bra j I-specific antibodies revealed the amount of Bra j I protein to be reduced or absent in seed extracts of selected lines. Removing an allergenic determinant from mustard is an important first step towards the development of safer food crops.

AB - Food allergies are a major health issue worldwide. Modern breeding techniques such as genome editing via CRISPR/Cas9 have the potential to mitigate this by targeting allergens in plants. This study addressed the major allergen Bra j I, a seed storage protein of the 2S albumin class, in the allotetraploid brown mustard (Brassica juncea). Cotyledon explants of an Indian gene bank accession (CR2664) and the German variety Terratop were transformed using Agrobacterium tumefaciens harboring binary vectors with multiple single guide RNAs to induce either large deletions or frameshift mutations in both Bra j I homoeologs. A total of 49 T 0 lines were obtained with up to 3.8% transformation efficiency. Four lines had large deletions of 566 up to 790 bp in the Bra j IB allele. Among 18 Terratop T 0 lines, nine carried indels in the targeted regions. From 16 analyzed CR2664 T 0 lines, 14 held indels and three had all four Bra j I alleles mutated. The majority of the CRISPR/Cas9-induced mutations were heritable to T 1 progenies. In some edited lines, seed formation and viability were reduced and seeds showed a precocious development of the embryo leading to a rupture of the testa already in the siliques. Immunoblotting using newly developed Bra j I-specific antibodies revealed the amount of Bra j I protein to be reduced or absent in seed extracts of selected lines. Removing an allergenic determinant from mustard is an important first step towards the development of safer food crops.

KW - Bra j I

KW - CRISPR/Cas

KW - food allergen

KW - mustard

KW - seed storage protein

KW - transformation

UR - http://www.scopus.com/inward/record.url?scp=85120429845&partnerID=8YFLogxK

U2 - 10.1111/tpj.15584

DO - 10.1111/tpj.15584

M3 - Article

C2 - 34784073

VL - 109

SP - 649

EP - 663

JO - Plant Journal

JF - Plant Journal

SN - 0960-7412

IS - 3

ER -