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 Nitrogen Use Efficiency (NUE). We also predict that similar genes will influence NUE in tomato as in Arabidopsis.

I(A) Induced targeted mutations in key genes that regulate N responsiveness in plants (Arabidopsis)

1. Mutations in feed-forward loops (FFL) components (Arabidopsis): We have designed and tested a suite of CRISPR/Cas to induce mutations in multiple core transcription factors (TFs) previously identified by the Brady lab as mediating the N transcriptional network. Multiplexed constructs have been designed to target four sets of transcription factor genes that function in FFLs. For each of these FFLs, we have designed five six different constructs to maximize the chances of obtaining the desired mutations. We initially tested constructs in transient protoplast assays before transforming into Arabidopsis plants using established floral-dip methods. Transgenic lines are currently being selected and genotyped for mutations at the target loci. Progeny seed will be collected and used for further genotypic, phenotypic and gene-expression analyses.

2. Monitoring nitrogen signal output (Arabidopsis): 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, we have transformed Arabidopsis plants with a luminescent:fluorescent fusion reporter-protein already in use within our lab driven by previously characterized NIR1 and NRP promoters. We have also established a hairy root transformation system for Arabidopsis and are currently regenerating hairy roots with the same fusion-reporter constructs. This will enable us to conduct rapid assays to assess changes in root morphology in response to nitrogen availability.

3. Validation of transcription factor binding sites in Arabidopsis promoters: To date, we have identified candidate binding sites in the promoters of the genes of interested using existing sequencing datasets (e.g. DAP-seq and DNAseI-seq). We are progressing two approaches to characterise candidates: (a) in vitro DNA-protein binding assays (b) expression analysis.
For strategy (a) we have designed and constructed plasmids to express each of the TF-proteins in cell-free and bacterial systems. We are currently working on protein expression and protein-DNA interaction assays. For strategy (b) we have designed and built plant expression constructs for each TF as well as a reporter construct in which the promoter of each TF gene is fused to a luminescent reporter (nanoluc). We have combinatorically co-expressed each reporter construct with each TF construct in Arabidopsis protoplasts. We have confirmed the function of some TFs as transcriptionally activators or repressors of downstream gene. These data confirm the existing yeast-one-hybrid data from the Brady lab. We are now proceeding to repeat these assays using promoters in which the candidate binding sites have been disrupted.

II(B) Induce targeted mutations in key genes that regulate N responsiveness in plants (tomato) - This work has been carried out by our collaborating labs (the Brady and Segal labs), at the University of California, Davis whose project was initiated as part of this USDA/NSF-BBSRC funding scheme.

4. Implementation of LOOP system for genome editing in tomato: Several promoters (AtRPS5Apro, AtYAOpro, and PcUBQpro) to drive Cas9 expression in tomato were tested and targeting efficiencies were compared. Basta and Kanamycin selections were tested and both were functional in tomato hairy roots. This enables the transformation of two independent constructs within tomato.
5. Characterization of 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 N 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.
6. Monitoring nitrogen signalling output: We generated and tested several Nitrogen-signaling output markers in tomato by fusing GFP to the promoters of known nitrate-responsive genes.
7. Translation of the Arabidopsis nitrogen signalling network to tomato: We 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. We have also generated deletion mutants in two of these genes.
8. Characterization of upstream regulatory regions of tomato genes and inference of feedforward loops: We have characterized the upstream regulatory regions of the putative tomato orthologs to determine the potential overlap of transcription factor-mediated regulation by the integration of unpublished, in-house ATACseq data, and identification of putative binding sites.

II Develop and apply tools for inducing persistent changes to the epigenetic status of plant genes
9. Our plan was to focus on genetic manipulations in the first period of the project and then epigenetic manipulations in Period 2. 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. We are also designing experiments to build and test a set of tools based on dCas9 designed to introduce persistent changes to the epigenetic status of plant genes.
Exploitation Route too early to say
Sectors Agriculture, Food and Drink

 
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 Synthetic Biology Expert Roundtable, Department for Business, Energy and Industrial Strategy
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
 
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 Scientific Advisory Board for Cluster of Excellence on Plant Sciences (CEPLAS), a joint initiative of Heinrich Heine University Düsseldorf (HHU), University of Cologne (UoC), Max Planck Institute for Plant Breeding Research Cologne (MPIPZ) and Forschungszentrum Jülich (FZJ). 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Member of the Science Advisory Board for the Cluster of Excellence on Plant Sciences (CEPLAS), a joint initiative of Heinrich Heine University Düsseldorf (HHU), University of Cologne (UoC), Max Planck Institute for Plant Breeding Research Cologne (MPIPZ) and Forschungszentrum Jülich (FZJ).
Year(s) Of Engagement Activity 2019,2020
URL https://www.ceplas.eu/en/home/