18 BTT EAGER - Engineering complex traits using targeted, multiplexed genetic and epigenetic mutagenesis

Lead Research Organisation: Earlham Institute
Department Name: Research Faculty

Abstract

Complex plant traits that respond to changes in the environment are regulated by large suites of genes. For example, the depletion of Nitrogen in the soil alters the levels and patterns of expression of large numbers of genes, affecting features such as root development, growth rate and flowering time. This has a considerable impact on the way that plants grow and develop and can lead to reductions in crop yields. Genetic variations are for crop improvement by selecting plants with specific variants associated with desirable traits for inclusion in breeding programs. To date, most focus has been on creating and exploiting genetic variations in the coding regions of genes. In this project we will focus on developing tools to create genetic and epigenetic variation in the non-coding regulatory sequences. Our aims are to introduce mutations that reprogram responses to environmental nitrogen availability. These will contribute new tools and approaches for the application of genome engineering to crop improvement and will also provide new knowledge about how variation in non-coding regulatory regions can be exploited to breed or engineer complex traits in crops.

Technical Summary

Genetic variations within coding sequences have been heavily exploited for crop improvement. However, quantitative, complex traits that respond to changes in the environment are the result of genetic and epigenetic variation in the non-coding regulatory sequences of multiple genes. Systems analyses of transcriptional networks have enabled the identification of suites of genes that coordinate network responses, shaping complex phenotypes. For example, a plant's environmental nitrogen status is coordinated by multiple factors interacting combinatorially. Such advances have coincided with the development of molecular tools for targeted genome engineering. Here, we propose to apply our expertise in genome engineering, systems and synthetic biology to engineer a complex trait. We will develop genome engineering technologies for inducing multiplexed mutations in coding and non-coding genic regions as well as epimutations in non-coding regions. We will apply these tools to create mutations in the genes that coordinate large-scale transcriptional responses to environmental nitrogen availability. Our work will also test if genetic factors recently identified in Arabidopsis also influence nitrogen use efficiency in tomato.

Planned Impact

The aim of this project is to develop genome engineering technologies for inducing multiplexed mutations in both coding and non-coding genic regions and epimutations in non-coding regions. Their functional outcome will be demonstrated by manipulating the expression of genes that coordinate response to environmental Nitrogen (N) status. Our work will also test if genetic factors recently identified in Arabidopsis also influence nitrogen use efficiency in tomato. This project is intended to develop new tools and approaches for engineering crops that are resilient to stress. While most genome engineering applied to plants has aimed to mutate coding sequences, we also aim to increase genetic variation in non-coding sequences as well as to introduce changes to the epigenetic status of genes. We will ensure that and novel tools, methods and experimental approaches are communicated and disseminated. We will also assess our engineered plants for increased growth and, if observed, progress further experiments to confirm our findings. During the project, we will engage and communicate with the public as well as with agrobiotech and breeding industries.

Publications

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Description The goal of this project was to apply genome engineering technologies to induce multiplexed mutations in both coding and non-coding genic regions to manipulate plant responses to changes in the availability of nitrate. We also aimed to develop tools for introducing
epimutations in non-coding regions. Our central hypothesis was that modulating the sequences of genes that regulate plant nitrate responsiveness would result in largescale changes in gene expression in response to nitrate and, consequently, changes to plant growth. In addition, we aimed to investigate if genetic factors recently identified in Arabidopsis also influence N-use efficiency in tomatoes. To date, we have completed the following objectives:

1. We designed and tested a suite of genome-editing constructs to induce mutations into multiple core transcription factors (TFs), previously identified as mediating the N transcriptional network. Constructs were designed to introduce mutations into one or more genes that comprise three different feed-forward loops within the network. For each set of genes, we designed six different constructs with different iterations which allowed us to identify the most efficient components and genetic architectures for our constructs. We tested the function of constructs in transient protoplast assays and subsequently delivered them into Arabidopsis plants using established floral-dip methods. We then identified lines with the desired mutations. We are in the process of growing lines in different nitrate conditions to quantify the effects of plant growth as well as changes to gene expression. Although we have yet to analyse all plant lines, we have demonstrated our ability to introduce multiplex mutations and confirmed our initial hypotheses that modulating the sequences of genes that regulate plant nitrate responsiveness results in largescale changes in gene expression in response to nitrate and, consequently, changes to plant growth.
2. To investigate how the nitrate responsive gene-regulatory network functions, and to understand the changes caused by disrupting the expression of genes, we conducted a series of experiments to investigate interactions between transcription factor proteins and their target genes within the network. This has been done using (i) a new plate-based assay to quantify protein-DNA binding affinity developed in our lab (ii) quantifying changes to expression levels of candidate target genes following transient expression of specific transcription factors and (iii) co-expression of transcription factors with reporter genes fused to target regulatory DNA. This has enabled us to gain a nuanced understanding of the interactions within the network and to identify specific binding sites for each transcription factor within its target genes.
3. To determine if genetic variation in non-coding regulatory regions of transcription factor genes changes the expression dynamics of the transcriptional network, we designed base-editing constructs to introduce mutations in specific transcription-factor binding motifs.
4. At UC Davis our partners have developed tools for epigenetic editing in plants. Tools for removing DNA methylation were successfully tested in Arabidopsis. We received these tools into our lab and have designed constructs to write methylation at target genes in the N-responsive network. These have been delivered to plants.
5. At UC Davis, our project partners have evaluated the use of several promoters for optimal genome editing in tomato hairy roots. They have also characterized the tomato root nitrogen signalling response: The root system architecture, shoot/root ratio, and expression patterns of potential nitrogen response genes were monitored. Marker genes for tomato nitrogen status were identified. They then identified the most likely tomato orthologs of the Arabidopsis N-responsive genes using pre-existing cell type-resolution tomato root gene expression profiling data and identifying genes with the most expression similarity to their Arabidopsis counterparts. They generated hairy roots cultures with equivalent mutations in the tomato homologues of those being studied in Arabidopsis. To analyse these lines, they developed a novel assay to investigate the effect of genetic changes to the expression on nitrogen status genes. Analysis of edited lines revealed that while some genes in the network are conserved between the two species, there are some differences.
6. Much of the work in this project was underpinned by the ability to rapidly construct different combinations of multi-gene constructs. To enable this, we successfully shared and implemented the LOOP cloning system that we previously co-developed in a recent project with project partners at UC Davis. The use of this system enabled us to share and reuse standardized DNA parts reducing effort across the project as well as creating a set of parts that can be used by other laboratories in the future.
Exploitation Route All tools developed in this project for editing the coding and non-coding regions of plant genes are widely applicable and will be made available to the research community where they can be deployed in other projects.

Our assays for investigating protein-DNA relative binding affinities will be useful to other research scientists that study gene regulation and networks.

Knowledge of the specific interactions within the nitrate-responsive regulatory network can be used to build accurate models, which can be used to inform future engineering strategies for improving plant nutrient use efficiency.
An understanding of the tomato nitrate-responsive network can be used to inform strategies to modulate tomato nitrate use efficiency.
Sectors Agriculture, Food and Drink

 
Description Advised the Parliamentary Office of Science and Technology on the content of a new POSTnote on gene editing in agriculture
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
URL https://post.parliament.uk/research-briefings/post-pn-0663/
 
Description Engagement with All-Party Parliamentary Group on Agriculture and Food for Development: Implications of the 2018 European Court of Justice ruling on genome editing
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Engagement with the All Party Parliamentary Group for Science and Technology in Agriculture. EI supported this APPG's amendment to the Agriculture Bill in Summer 2020 when the Bill was going through the House of Lords. The amendment sought to give the DEFRA Secretary of State power to introduce a new regulatory regime to allow the commercial use of gene editing for crops for agricultural purposes. EI orchestrated a cross-NRP advocacy effort to brief members of the House of Lords to support the amendment and signed the NIAB letter to DEFRA Secretary of State articulating our support for the amendment. The impact of our work helped to bring about the Government's announcement of a public consultation with a view to primary legislation to establish a new regulatory regime to enable gene editing.
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
 
Description Expert member of the plant synthetic biology working group for the European Food Standards Agency (EFSA)
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
 
Description Policy Round Table on Synthetic Biology, UK Cabinet Office
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
 
Description Science and Technology Committee, House of Commons - implications to the UK of the 2018 Euporean Court of Justice ruling on Genome Editing
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Synthetic Biology Expert Roundtable, Department for Business, Energy and Industrial Strategy
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
 
Description The Earlham Institute hosted a policy roundtable discussion on new regulation for genetic technologies with 15 members of the DEFRA policy team
Geographic Reach National 
Policy Influence Type Participation in a national consultation
 
Description Genetic determinants of transcriptional stochasticity 
Organisation Biochemistry and Plant Molecular Physiology
Country France 
Sector Public 
PI Contribution Collaborative experiments to investigate the role of transcription-factor-DNA interactions in stochastic gene expression
Collaborator Contribution Collaborative experiments to investigate the role of transcription-factor-DNA interactions in stochastic gene expression
Impact Ongoing
Start Year 2021
 
Description "An edit for Good?" Popular Science article for lab News 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Article for the online magazine, Lab News: "Gene editing could allow incredible crop improvements, with the potential to reduce the harmful impact of agrochemicals on biodiversity while boosting yield. However, the recent European Court of Justice ruling that gene editing be regarded as the same as GM poses a huge barrier to farmers and hamstrings European science. So, should the UK embrace gene-edited crops post-EU?"
Year(s) Of Engagement Activity 2019
URL http://www.labnews.co.uk/article/2024846/an_edit_for_good
 
Description Article: 'Should we genetically edit the food we eat?' The Conversation. 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Article co-written with an editor and social scientist and published at 'The Conversation'. This article is part of The Conversation's 'Head to Head' series, which feature academics from different disciplines chewing over current debates.
Year(s) Of Engagement Activity 2021
URL https://theconversation.com/should-we-genetically-edit-the-food-we-eat-we-asked-two-experts-162959
 
Description Blog post "N is for nitrogen: how can we make our crops more sustainable?" 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Blog post "N is for nitrogen: how can we make our crops more sustainable?"
Year(s) Of Engagement Activity 2021
URL https://www.earlham.ac.uk/articles/n-nitrogen-how-can-we-make-our-crops-sustainable
 
Description Blog post - 'Unlocking power of gene editing to protect the natural environment' 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Blog post - 'Unlocking power of gene editing to protect the natural environment'
Year(s) Of Engagement Activity 2021
URL https://www.earlham.ac.uk/newsroom/unlocking-power-gene-editing-protect-natural-environment-earlham-...
 
Description Blog post: New legislation granted to progress plant gene editing in UK 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Blog post: New legislation granted to progress plant gene editing in UK
Year(s) Of Engagement Activity 2022
URL https://www.earlham.ac.uk/newsroom/new-legislation-granted-progress-plant-gene-editing-uk
 
Description Interview with iNews 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Interview with iNews Science and Environment correspondent. This resulted in the article: "Scientists welcome Government plans to lift ban on gene editing in agriculture" published March 16, 2021.
Year(s) Of Engagement Activity 2021
URL https://inews.co.uk/news/science/scientists-welcome-government-plans-to-lift-ban-on-gene-editing-in-...
 
Description Neo.Life - 25 Visions for the Future of our Species (Book) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Contributed a book section to Neo.Life - 25 Visions for the Future of our Species (Book). Editors: Jane Metcalf and Brian Bergstein
Year(s) Of Engagement Activity 2020
URL https://neo.life/visions/
 
Description Policy seminar on gene editing 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact A webinar on "Making crops for the future: Innovative plant science explained" was hosted by BASF, VIB, Ghent. The majority of attendees were advisors for MEPs, students and scientists from industry. There was significant discussion on the technical differences between genome editing and other plant breeding techniques, and how these are regulated.
Year(s) Of Engagement Activity 2020
URL https://agriculture.basf.com/global/en/media/public-government-affairs/agri-science-policy-events.ht...
 
Description The Crop it Like it's Hot Podcast. A collaboration between Arable Farming magazine and The CropTec Show. Episode 11, 'Gene edited crops - quick fix or sustainable solution?' 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact A podcast aimed at UK growers focussed on DEFRA's public consultation on gene editing. We contributed an interview about what gene editing is, what it offers plant breeders, and what new traits might be developed.
Year(s) Of Engagement Activity 2021
URL https://www.croptecshow.com/podcasts/