FAQs: Planned field trial of CRISPR-Cas9 Gene-edited Brassica oleracea

Researchers at the John Innes Centre have applied to DEFRA to grow a small plot (25 m2) of CRISPR-Cas9 gene-edited Brassica oleracea in the spring and summer of 2019.

Here we explain why this field trial is important and how it may contribute to our understanding of the regulation of sulphur metabolism in this agronomically important crop.

Why CRISPR Brassica?

The Brassicaceae family contains many agronomically important crops, including oilseed rape and cruciferous vegetables. Cruciferous vegetables can be found in a wide variety morphotypes and have been consumed the world over for many years, having been bred and selected for their distinct flavor properties. The distinct flavor of Brassica vegetables is partially due to sulphur-containing metabolites, including the secondary metabolites known as glucosinolates.

Glucosinolates are produced exclusively by plants of the order Brassicales (also referred to as Capparales) in order to deter herbivory (Mithen et al., 2010). The production of these sulphur-containing secondary metabolites is of economic significance due to their putative health-promoting abilities upon human consumption.

The Myb28 gene has been repeatedly and independently well characterized as a vital regulator of aliphatic glucosinolate biosynthesis across the Brassica genus (Augustine et al., 2013; Kim et al., 2013; Liu et al., 2016; Seo et al., 2016).

It would be useful to understand the consequences of a knock out of this gene via CRISPR on sulphur metabolism and the production of glucosinolates in Brassica oleracea. The plants in the trial have modified Myb28 sequence in order to remove gene function so that we may characterize the effect this genetic disruption has on the production of these sulphur compounds in a commercial environment.

What has technically been done to the Brassicas?

Researchers at the John Innes Centre have recently developed protocols in order to utilise the recent breakthrough technology in genetics, CRISPR-Cas9, particularly in agronomically important crops such as brassicas and barley (Lawrenson et al., 2019; Lawrenson et al., 2015). These techniques allow an easier and more feasible route to direct molecular characterisation of genes, with historical techniques in gene-editing having often favoured model organisms.

The CRISPR Brassica oleracea lines were produced via Agrobacterium-mediated transformation of a bacterial plasmid containing the Cas9 (often referred to as molecular scissors) and guide sequences designed to specifically target the Cas9 ‘cutting’ activity to the gene of interest, in this case Myb28.

This transformation involved the use of 4-day old cotyledonary explants, as described in (Sparrow et al., 2006). The resulting plants were screened for the presence of mutations specifically in the target gene, with plants showing evidence of gene-editing being kept for seed.

Screening of subsequent generations has led to the Brassica oleracea plants to be used in this trial, which have a disruption in their gene sequence, leading to a proposed non-functional protein.

In addition, the generation being studied show no current evidence of the transgene or Cas9, which appears to have segregated out, leaving a plant with the desired mutation without the remaining aspects of the vector. As a result, the plants contain a mutation in a gene to stop it from working. The nature of this change is exactly as might have occurred naturally, and which occurs frequently in nature. There is no current evidence of ‘foreign’ DNA in the brassica plants.

Why a field trial?

Fundamentally, the aim of this trial is to better characterise the role of a gene, known as Myb28, in regulating sulphur metabolism, specifically the accumulation of aliphatic glucosinolates, in field-grown Brassica oleracea. Brassica plants of this type, when grown under glasshouse conditions, produce almost undetectable levels of these compounds, therefore this trial is required in order to better imitate the commercial interaction between these compounds and their environment and ultimately how this transcription factor Myb28 may mediate this interaction.

Field evaluation of these traits allows for a better understanding of how these crops plants function.

Is a field trial safe for the environment?

Yes. Brassica oleracea varieties such as cauliflower, kale, broccoli and cabbage are widely grown in the UK, and the main risks to consider are the spread of pollen and spread of seed. The Brassica plants in this field trial will be surrounded by a border of Brassica napus (or oilseed rape) which will act as a physical barrier to potential pests as well as a barrier to prevent pollen escape.

A zone of 20 meters will be left between the plants in this field trial and other test plots of non-GM Brassica to avoid the spread of pollen, with plants capable of cross-pollination being removed from the surrounding area through treatment with a pesticide if necessary.

The Brassica plants will be grown in a cage, used in previous field trials by the John Innes Field Experimentation team.

Prior to flowering, during the bud stage, the majority of plants will have their complete inflorescences harvested for genetic and metabolomic analyses. The remaining plants (no more than ten) that will be left to flower will be secured within pollen-proof bags to prevent the release of pollen to surrounding areas.

Upon completion of the trial all the Brassica plants will be destroyed, to the satisfaction of the regulator, and the land will be monitored for any residual Brassica plants.

Who will benefit from the CRISPR Brassica?

This trial is being proposed purely for exploratory purposes and no commercial or future commercial use is proposed at this time

The trial will help to mediate fundamental research in how sulphur metabolism is regulated in Brassica oleracea. This crop is widely consumed the world over and is often implicated for its potential health-promoting activities, due to the accumulation of the sulphur metabolites being studied in this trial, including glucosinolates. This trial will contribute a better understanding of the accumulation of these metabolites in this agronomically important crop, in the context of a commercial environment.

Who has funded the research?

The research has been funded by the UK Biotechnology and Biological Sciences Research Council.

References

• Augustine, R., Majee, M., Gershenzon, J., and Bisht, N.C. (2013). Four genes encoding MYB28, a major transcriptional regulator of the aliphatic glucosinolate pathway, are differentially expressed in the allopolyploid Brassica juncea. Journal of Experimental Botany 64, 4907-4921
• Kim, Y.B., Li, X., Kim, S.J., Kim, H.H., Lee, J., Kim, H., and Park, S.U. (2013). MYB Transcription Factors Regulate Glucosinolate Biosynthesis in Different Organs of Chinese Cabbage (Brassica rapa ssp pekinensis). Molecules 18, 8682-8695.
• Lawrenson, T., Hundleby, P., and Harwood, W. (2019). Creating Targeted Gene Knockouts in Brassica oleracea Using CRISPR/Cas9. In Plant Genome Editing with CRISPR Systems (Springer), pp. 155-170.
• Lawrenson, T., Shorinola, O., Stacey, N., Li, C., Østergaard, L., Patron, N., Uauy, C., and Harwood, W. (2015). Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome biology 16, 258.
• Liu, J., Hirani, A.H., Li, Z., Wu, C., McVetty, P.B., Daayf, F., and Li, G. (2016). QTL controlling glucosinolate content in seeds of’Brassica napus’ L. Australian Journal of Crop Science 10, 152.
• Mithen, R., Bennett, R., and Marquez, J. (2010). Glucosinolate biochemical diversity and innovation in the Brassicales. Phytochemistry 71, 2074-2086.
• Seo, M.-S., Jin, M., Chun, J.-H., Kim, S.-J., Park, B.-S., Shon, S.-H., and Kim, J.S. (2016). Functional analysis of three BrMYB28 transcription factors controlling the biosynthesis of glucosinolates in Brassica rapa. Plant molecular biology 90, 503-516.
• Sparrow, P.A.C., Dale, P.J., and Irwin, J.A. (2006). Brassica oleracea. In Methods in Molecular Biology, K. Wang, ed., pp. 417-426.