CRISPR-Cas9-mediated editing ofmyb28impairs glucoraphanin accumulation ofBrassica oleraceain the field

Abstract

We sought to quantify the role of MYB28 in the regulation of aliphatic glucosinolate biosynthesis and associated sulphur metabolism in field-grown B. oleracea with the use of CRISPR-Cas9-mediated gene editing technology. We describe the first characterised myb28 knockout mutant in B. oleracea, and the first DEFRA-regulated and approved CRISPR field trial in the UK to require compliance with the 2001/18 EU GMO directive. We report that knocking-out myb28 results in downregulation of aliphatic glucosinolate biosynthesis genes and reduction in accumulation of the methionine-derived glucosinolate, glucoraphanin, in leaves and florets of field-grown myb28 mutant broccoli plants. There were no significant changes to the accumulation of sulphate, S-methyl cysteine sulfoxide and indole glucosinolate in leaf and floret tissues.

Sustainable Use of Bioactive Compounds from Solanum Tuberosum and Brassicaceae Wastes and by-Products for Crop Protection—A Review

“…Furthermore, a genetic linkage map and QTL analysis revealed seed quality traits like erucic acid content and GSL in Sinapis alba L., providing useful tools for breeding strategy mediated by molecular markers associated with GSLs components [ 216 ]. Recently, a CRISPR/Cas9-mediated editing of MYB28 gene in Brassica oleracea has been achieved by Neequaye et al [ 201 ], and although the paper has not been subjected to peer review process so far, this result shed light on the opportunity that new biotechnological approaches could be applied to modify the content of GSLs in planta .…”

A Field Day for Gene-Edited Brassicas and Crop Improvement

“…Because of (or perhaps despite) all the mentioned reasons, a new article by Neequaye et al 3 in Lars Østergaard's group at the John Innes Centre, in Norwich, United Kingdom, in this issue of The CRISPR Journal should be applauded, for the authors have opted to take that road less traveled and evaluate their CRISPR-Cas9 edited Brassica oleracea plants in the field.…”

“…2 To date, most efforts have focused on spurious editing at unintended sites in the genome. A wealth of literature is dedicated to identifying 3,4 and mitigating [5][6][7][8][9][10] these off-target edits. Recent observations have extended spurious editing events to the target site itself, 11 again prompting careful examination of editing outcome.…”

Brassica oleracea Transformation

“…alboglabra (A12DHd) crossed with B. oleracea ssp. italica (Green Duke GDDH33) mapping population [19]. AG DH1012 is a spring type and self-compatible.…”

“…The use of transformation/CRISPR as a tool for genetic improvement in Brassica has recently been reviewed [18]. The AG DH1012 B. oleracea spring genotype, detailed in this chapter, has a broccoli type phenotype and was recently used in a study to generate MYB28 knock outs, which were subsequently grown under field conditions and were the first CRISPR/Cas9 edited plants to enter field trials in the UK, regulated under the EU GMO Directive [19].…”