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

authored by

Juvenal Assou, Dingbo Zhang, Kristian D R Roth, Stephan Steinke, Michael Hust, Thomas Reinard, Traud Winkelmann, Jens Boch

Abstract

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.

Organisation(s)

Woody Plant and Propagation Physiology Section
Institute of Horticultural Production Systems
Section Plant Biotechnology
Institute of Plant Genetics

External Organisation(s)

Technische Universität Braunschweig

Type

Article

Journal

Plant Journal

Volume

109

Pages

649-663

No. of pages

15

ISSN

0960-7412

Publication date

29.01.2022

Publication status

Published

Peer reviewed

Yes

ASJC Scopus subject areas

Genetics, Plant Science, Cell Biology

Electronic version(s)

https://doi.org/10.1111/tpj.15584 (Access: Open)