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

10 25 50
 
Description The main goal of this project was to develop genome engineering technologies for inducing multiplexed mutations in both coding and non-coding genic regions and epimutations in non-coding regions. We planned to test functional outcomes by manipulating the expression of genes that coordinate response to environmental Nitrogen (N) status. Our work also aimed to test if genetic factors recently identified in Arabidopsis also influence N-use efficiency in tomato. Our hypothesis was that knocking out or modulating the expression levels of TFs that regulate N responsiveness will result in changes in expression across the gene network resulting in plants with altered NUE. We also predict that similar genes will influence N use efficiency tomato as in Arabidopsis.

In the UK, we have 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. Multiplexed constructs have been designed to target the genes that comprise three different feed-forward loops within the network. For each set of genes, we have designed six different constructs with different iterations to allow us to identify the most efficient components and architecture. We initially tested the function of constructs in transient protoplast assays and subsequently delivered them into Arabidopsis plants using established floral-dip methods. In the last year, we have primarily focussed on identifying plant lines with the desired mutations. Due to Covid-19-related delays, this is still ongoing. In addition, we have transformed plants with reporter constructs to create control lines that express luminescent and fluorescent proteins in response to changes in available nitrate. To do this, have transformed Arabidopsis plants with a luminescent:fluoresecent fusion reporter driven by the previously characterized promoters.

A second aim was to determine if genetic variation in non-coding regulatory regions of genes in the genes of TFs that regulate Nitrogen responsiveness will change expression across the gene network. Our aim is to mutate transcription factor recognition sites and thus disrupt the flow of information across the network. To do this, we have first progressed a series of experiments to validate predicted interactions between transcription factor proteins and DNA targets. This is being done using a new quantitative luminescence assay developed in our lab as well as by quantifying changes to expression levels of candidate target genes following transient expression of specific transcription factors. To date, we have confirmed several key interactions within the proposed network. We have also designed genome-editing constructs to mutate key regions and motifs of the promoters.

Another goal of this project was to develop tools for epigenetic editing in plants. We have designed and assembled several constructs to the epigenetic status of key genes within the network.

Finally, much of the work in this project is 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 with project partners at UCDavis who are progressing parallel experiments in tomato. This enables us to share and reuse standardized DNA parts such as Cas9 and interchanging species-specific modules such as sgRNAs and regulatory elements.

At UC Davis, our project partners have: Implemented the LOOP system for genome editing in tomato and evaluated the use of several promoters for optimal genome editing in tomato hairy roots. This enabled the transformation of two independent constructs within tomato. They have also characterized the tomato root nitrogen signalling response: Tomato M82 seeds were grown on media supplemented with 0, 0.5, 1, 5, 10, and 20mM of KNO3 as the only Nitrogen source. The RSA, shoot/root ratio, and expression patterns of potential nitrogen response genes were monitored. A marker gene for tomato nitrogen status was identified. They then generated and tested several Nitrogen-signalling output markers in tomato by fusing GFP to the promoters of known nitrate-responsive genes.

They have identified the most likely tomato orthologs 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 are currently generating hairy roots cultures with mutations in the tomato homologues of those being studied in Arabidopsis.

Finally, they have designed and assembled several constructs to the epigenetic status of key genes within the network. In past work, it was shown that tethering the non-catalytic SUVH9 protein to an engineered zinc finger (ZF) DNA-binding protein was sufficient to recruit components of the RNA-directed DNA methylation pathway (RdDM) system to a hypo-methylated allele of the FWA gene, resulting in hyper-methylation and rescue of the normal early flowering time. Our collaborators have obtained the ZF-SUVH9 construct and the fwa-4 hypomethylated epiallele and are in the process of reproducing these published results as a positive control.
Exploitation Route too early to say
Sectors Agriculture, Food and Drink

 
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 "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 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/