Professor Wendy Harwood leads a team of researchers and technicians at the John Innes Centre who have expertise in tissue-culture based plant transformation techniques (BRACT- Biotechnology Resources for Arable Crop Transformation).
Transformation is often considered a dark-art of plant science, as history informs that it requires an established set of protocols that have been well-tested for local experimental conditions. It takes significant time and expertise to set up an effective and reliable transformation pipeline, which is why few places around the UK undertake this type of work. These challenges can provide a significant bottleneck in the establishment of high-throughout transformation platforms.
The BRACT expertise in transformation was aligned with gene editing technology to successfully gain UKRI-BBSRC Bioinformatics and Biological Resources Funding (BBR) funding in 2016 with a grant entitled ‘Targeted gene knockouts in crops using RNA-guided Cas9 nucleases’. Professor Harwood shares Co-I status on this grant with Dr Nicola Patron who is an expert on synthetic biology and genome editing based at the Earlham Institute.
Expanding UK capability in gene editing of crops
This BBR funding is absolutely community-facing and included the provision for 50 genes, selected from open applications, to be targeted by CRISPR-Cas9 gene editing. Initially successful applicants would benefit from BRACT expertise in guide RNA (gRNA) design after which the optimised gRNAs and Cas9 would then be introduced into either barley or Brassica oleracea. However, the success of the BRACT service meant that over 60 genes were ultimately selected for targeted knock-out and in the second round of applications the available crops were expanded to include wheat, Brassica napus and tomato.
This grant allowed users throughout the UK to take advantage of expertise in gene editing and transformation, which is beyond the capabilities of most individual research departments. The grant also included a requirement to provide training for new users and a training event was successfully delivered between July 10-12th at the JIC. On days 1 and 3, 12 successful applicants worked with the BRACT team to learn how to design CRISPR guides, assemble constructs, screen edited plants and perform the basics of plant transformation. In between these two days was a day of scientific talks that included talks from successful community applicants as well as updates on the latest technical developments at BRACT. The day 2 workshop was attended by 62 participants from across the UK. The demand for training was significantly higher than the number of spaces available, with the course being 3x oversubscribed. Hopefully this may encourage the course organisers to consider running similar training events again.
Demonstrating a clear community need.
During the two rounds of applications, researchers from 43 UK research groups across 26 institutions applied to have one or more genes of interest targeted by this resource. Due to high demand ultimately 61 genes were selected for targeting from a total of 126 requested genes. Multiple guide RNAs were designed and primary transformation conducted at BRACT before transgenic and potentially edited plants were sent out to users. The users then screen these plants for Non-homologous end joining (NHEJ)-editing events and, if needed, identified transgene-free germplasm. BRACT provided help with screening and growing on the selected plants as required.
Success Stories
Mikhaela Neequaye presented the most complete study of those that have used the BRACT facility through this BBR funding. She works with Richard Mithen, formally from the Quadram Institute and Lars Ostergaard at the JIC on the Brassica oleracea MYB28 transcription factor, which is involved in glucosinolate biosynthesis. In collaboration with BRACT she has identified plants with edited MYB28 that show altered glucosinate levels and has even successfully gone through the onerous process of applying to DEFRA to conduct a field trial with these edited plants. An impressive output from a PhD project!
Kate Henbest is a PhD student who works with Lorraine Williams at the University of Southampton on the challenge of developing plants with altered capacity to transport zinc. She targeting two loci encoding bZIP transcription factors in barley and had successfully identified the predicted double ‘CRISPR mutants’. In addition she also found a surprising triple mutant that also contained a mutation in a related bZIP family member. These multi-mutants were identified from a relatively small number of edited lines, which so far is a very satisfying result.
However the appearance of the triple mutant highlights that we have an incomplete understanding of the editing events that will occur after NHEJ and demonstrate the importance of users feeding back this information to BRACT. This will allow them to build a valuable database about the particular editing events observed by users. The handful of users who presented their research at the workshop documented both small deletions as well as single nucleotide substitutions and deletions.
Growing plants can be surprisingly challenging!
Early user feedback indicates that obtaining full-value from this BBR grant might rely on the extent of post-editing support that the BRACT team is able to provide (and how this might be financially supported). Some applicants had previously only worked with Arabidopsis, so after a successful application they will be confronted with the challenge of growing much larger (transgenic) plants that will require more space and different growth conditions. This might seem like a trivial problem but in order for the BRACT team to show that their project has been successful (in terms of follow-on publications or future grants) then this relies on end-users being able to correctly grow plants in order to obtain useful data. The BRACT team has been able to provide some advice on these issues without impinging on the time they must devote to other projects, but as is often the case, these additional requirements are often hidden time constraints that aren’t always factored into grant funding.
Somewhat related to these issues was a talk from Gustaf Degen from Lancaster University who had received Brassica oleracea plants with edits within a gene involved in photosynthesis. His progress in this project has been delayed as all of the edited plants died during a particularly (surprisingly) hot summer in Lancaster! Fortunately, the plants were saved by returning to the original tissue-culture and although this was a fluke event, it does rather highlight that users need to be able to grow the plants that they intend to work with!
Overcoming the challenges of HDR.
Tufan Oz had recently joined the Patron lab at the Earlham Institute but gave a talk about his previous research conducted in Florida. He worked with sugarcane and had successfully targeted the acetolactate synthase (ALS) gene using homology-directed repair (HDR). He explained that sugarcane provides an excellent model for HDR as the tissue-culture transformation frequency is very high.
One of the BRACT CRISPR-experts is Tom Lawrenson and he gave an informative update about his work aiming to undertake HDR in barley. HDR uses a ‘conventional’ gRNA/CRISPR system along with an additional DNA template with homology arms to a specific pre-selected region within the genome. Although HDR results in the insertion of a ‘foreign’ gene unlike conventional GMOs that rely on random insertion, HDR allows targeted insertion to a chosen place in the genome.
A particular challenge of using HDR is ensuring that there is a sufficiently high quantity of the DNA template in the vicinity of the Cas9 cut site. Poor transformation frequencies will also be an impediment to this work as with fewer transformations there is less chance of obtaining a line with sufficient high levels of the template. Tom reported that overall this means that successful HDR occurs 10-100x times less efficiently than NHEJ.
A major strength of this BBR grant is that it complements other funded work [wh(1] aiming to troubleshoot challenging protocols (such as HDR) precluding the need for replicated efforts in labs around the country. In these HDR experiments Tom is attempting to directly knock-in a N-terminal mCherry fusion and his primary advice is to ensure that you use very efficient gRNAs. Due to the low efficiency of HDR in order to obtain a correctly edited plant a researcher has the choice to either a) look at more primary transformants, b) to use a viral vector to improve expression or c) to screen a lower number of transformants over multiple generations. Tom has experimented with viral-derived components but found that this wasn’t necessary for successful HDR. As with NHEJ, the requirements for efficient HDR will vary on a plant-to-plant basis but the BRACT team are working out the basics so that the entire community will benefit from using this powerful technique in the future.
Future technologies
The latter stages of the workshop focused on future technologies that could be of use to the community. The BRACT team discussed their recent work on:
1. Use of Cpf as an alternative to Cas9 (it doesn’t work as well in their hands)
2. On their testing of base-editing and epigenome editing techniques (work in progress)
3. Strategies to obtain multiple knock-outs from a single transformation (use different promotors to ensure even expression of all gRNAs in a single construct).
Workshop participants were invited to suggest what they needed to improve their research using CRISPR-based technologies. The consensus amongst those few people I talked to was that more hands-on training would be very useful and that nothing is too simple to be taught. This type of training requires a significant commitment in time and space so there needs to be a community discussion around the mechanisms that could be used to enable these opportunities. CRISPR-based gene editing techniques are clearly game-changing technologies but, as with anything, without correct use they can take a lot of time for little reward.
During discussions Professor Harwood gave a comment on the ‘Freedom To Operate’ study that they have commissioned as part of this BBR grant. This suggests that they will have no difficulty using CRISPR-based technology for projects working with academic applicants. Interestingly it was positive to learn that whereas there may be complications when they work with for-profit companies, it should be possible for those companies to use the CRISPR technology without having to pay a prohibitive licensing fee. However as the legal battles over the CRISPR-patent have not yet been resolved any opinion given in this area might be liable to change.
The day of presentations ended in traditional John Innes Centre fashion with some locally brewed beer and wine on the terrace in the summer sun! The three-day workshop / training course then concluded for the trainees with the third day allowing hands-on experience of tissue-culture transformation techniques. Hopefully the training course participants will take this knowledge back to their home institutions in order to allow more researchers to master one of the dark-arts of plant science!
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