18-BTT Clean genome editing through the use of nonintegrating T-DNA technology
Lead Research Organisation:
University of Leeds
Department Name: Ctr for Plant Sciences
Abstract
Genetic modification of crop species is the key to both food security and sustainable agriculture. The advent of CRISPR/Cas technology has provided a great advance in our ability to engineer genomes, but barriers remain to the routine employment of these methods in the most important agricultural species. This proposal addresses the most significant problem in engineering crop plants, that genome modification is associated with untargeted and potentially mutagenic integration of the machinery used to edit the genome. This is problematic due to the increased screening required to identify targeted transformants against the high background of random integrations. In addition, for commercial use the synthetic constructs must be eliminated from the genome in a process that can be lengthy and expensive for many crops. This project will develop a clean genetic engineering methodology based on the suppression of random transgene integration. This technology builds on the identification of a DNA Polymerase Theta (PolQ)-mediated pathway that is responsible for the majority of transgene integration events. We will suppress this pathway and investigate the effect on gene targeting frequencies. Proof of principle will be provided in Arabidopsis through targeted mutation of the ABI1 gene, resulting in the production of a dominant mutation that allows germination in the presence of abscisic acid. This work will be extended to Brassica to demonstrate the application of this technology to crop species. This project will significantly advance our ability to engineer crop genomes using a knowledge-based approach and informed by the applicants' considerable experience in plant transformation and DNA recombination mechanisms.
Technical Summary
Genetic modification of crop species is the key to both food security and sustainable agriculture. The advent of CRISPR/Cas technology has provided a great advance in our ability to engineer genomes, but barriers remain to the routine employment of these methods in the most important agricultural species. This proposal addresses the most significant problem in engineering crop plants, that genome modification is associated with untargeted and potentially mutagenic integration of the machinery used to edit the genome. This is problematic due to the increased screening required to identify targeted transformants against the high background of random integrations. In addition, for commercial use the synthetic constructs must be eliminated from the genome in a process that can be lengthy and expensive for many crops. This project will develop a clean genetic engineering methodology based on the suppression of random transgene integration. This technology builds on the identification of a DNA Polymerase Theta (PolQ)-mediated pathway that is responsible for the majority of transgene integration events. We will suppress this pathway and investigate the effect on gene targeting frequencies. Proof of principle will be provided in Arabidopsis through targeted mutation of the ABI1 gene, resulting in the production of a dominant mutation that allows germination in the presence of abscisic acid. This work will be extended to Brassica to demonstrate the application of this technology to crop species. This project will significantly advance our ability to engineer crop genomes using a knowledge-based approach and informed by the applicants' considerable experience in plant transformation and DNA recombination mechanisms.
Planned Impact
Impact Summary
Publications
Piskunen P
(2022)
Integrating CRISPR/Cas systems with programmable DNA nanostructures for delivery and beyond
in iScience
| Description | Gene targeting involves precise genome editing using a DNA template that is introduced into the cell (a transgene). The hosts DNA repair pathways use this template as a guide, coping this sequence into a region of homology in the hosts own DNA (genome).This project investigated gene targeting in the model plant Arabidopsis thaliana to determine whether it was possible to create desired changes in the plant genome without the unwanted integration of the gene editing machinery. The hypothesis was that blocking the random integration pathway would allow precise gene editing but would prevent stable integration of DNA introduced into the plant cell. A main method of creating genetically modified plants involves the use of the natural genetic engineer, the bacterium Agrobacterium tumefaciens. This is widely used in biotechnology, whereby the DNA that is passed from the bacterium to the plant, termed T-DNA, can be modified to create the desired changes, for example plant resistance to pests. In this process, the bacterium relies on host factors within the plant to integrate the DNA. Recently the major pathway for integration was identified as being mediated by a specialised DNA polymerase, termed PolQ. This project aimed to disrupt this pathway but expressing variants of PolQ that would interfere with its function and allow plant modification without the unwanted changes associated with current technologies. We successfully engineered and expressed a range of PolQ derivatives, although expression of the full length protein was toxic to agrobacteria. In order to test the efficacy of the PolQ derivatives, an assay for gene editing was developed. Initially, the approach was to engineer resistance to herbicides or the plant hormone abscisic acid as the products of gene editing. This would provide powerful assays to allow screening of rare events. A number of optimisations were made, generating a bank of genetic resources for future projects. However, the potential detection of rare events, which involved screening millions of seeds, did not result in isolating the desired plants. In the light of this, a novel assay was developed based on the detection individual cells that were products of successful gene editing, resulting in the production of Green Fluorescent Protein (GFP). This assay was amenable to high throughput quantification using automated fluorescent cell counting. This successfully identified the products of gene editing, which were also confirmed by confocal microscopy. Attempts to inhibit random integration using the PolQ variants proved difficult due to toxicity of these variants in bacterial cells and required further optimisation. The project has therefore successful established gene editing assays and enabled further work to optimise this technology. |
| Exploitation Route | A robust and accessible gene targeting assay will help improve this technology and in the development of improved crop varieties. In addition, the development of inhibitory PolQ derivatives will help positively influence the outcome of gene targeting events in plants. Taken together, the results from this project will help advance precise plant genome engineering |
| Sectors | Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology |
| Description | The project was used to develop an education resource as part of the University of Leeds International Summer School. A module was developed to introduce methods and experience of making transgenic plants, including gene editing techniques. |
| First Year Of Impact | 2020 |
| Sector | Education |
| Impact Types | Societal |
| Description | Application of nanotechnology to gene targeting in plants |
| Organisation | University of Leeds |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | This collaboration is supported by a joint BBSRC DTP studentship jointly supervised by Matteo Castronovo in Food Sciences at the University of Leeds. My group provides expertise and resources in plant transformation and analysis |
| Collaborator Contribution | The group of Dr Castronovo provides expertise and resources in nanotechnology based on folding DNA into higher order structures termed DNA origami |
| Impact | A review paper is in press |
| Start Year | 2020 |
| Description | Project application |
| Organisation | University of Leeds |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | This is a project proposal for the Pioneer award scheme (UKRI) and we provided expertise on seed biology and proteomics analysis |
| Collaborator Contribution | The partner at the University of Leeds (Matteo Castronovo) provided expertise in nanotechnology to develop a model system for DNA breaks |
| Impact | Pioneer expression of interest approved and a full application submitted |
| Start Year | 2022 |
| Description | Biology taster session |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Schools |
| Results and Impact | Greenhead College - lunchtime Biology taster session. Presentation on my PhD project to sixth form students. Positive feedback received, particularly from one student interested in DNA. |
| Year(s) Of Engagement Activity | 2022 |
| Description | Educational outreach |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Schools |
| Results and Impact | Educational Outreach Fellow with the Educational Engagement Department at the University of Leeds, we developed and present activities for school and college students to engage and inspire them in STEM, often with students from disadvantaged backgrounds. A'GM Debate' activity promotes development of 'soft skills' (public speaking, debate, nuance) in the students and was received very positively. |
| Year(s) Of Engagement Activity | 2021,2022 |
| Description | Invited speaker at conference |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | Invited speaker at International Symposium on Plant Cell Cycle and DNA Damage Responses 2022 |
| Year(s) Of Engagement Activity | 2022 |
| Description | Medicine - not just for Medics hosting school children at the University of Leeds |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Schools |
| Results and Impact | 'Medicine - not just for Medics' 4 hour lab ~15 year 10 students from a variety of schools came onto campus. DNA extraction from a strawberry + accompanying lecture explaining the structure of DNA and DNA in forensics. |
| Year(s) Of Engagement Activity | 2022 |
| Description | Reach For Excellence programme hosting school children at the University of Leeds |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Schools |
| Results and Impact | 2 x 2 hour sessions (Feb 22 and Feb 23) 'The importance of plant biology?' with a focus on GM crops as part of the Reach For Excellence programme - a programme where college (year 12) students from disadvantages schools come onto campus and choose talks in their area of interest to inspire them to do that subject at University. Good feedback from session lead saying that the students really enjoyed it and it was an interesting topic. One student said they were now very interested in studying biology at University after the session. General comments from students about enjoying the topic matter and getting the chance to practice presentations and group work, citing improved confidence and team working skills. Repeated this session online with the Linacre institute, July 2022, for ~15 students, and with Ashton College 6th Form for ~20 students. |
| Year(s) Of Engagement Activity | 2022 |
| Description | Schools visit/ discovery zone |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Schools |
| Results and Impact | Over 100 year 12 students visited the University and my group presented an activity based on genome editing. This raised awareness of techniques used in biotechnology and agriculture amongst the target audience |
| Year(s) Of Engagement Activity | 2020 |
| Description | tutoring |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Schools |
| Results and Impact | Tutoring - New College Pontefract (New Collaborative Teaching Trust) - extended periodic tutoring of a student in their CREST Award project in 2022 about the application of gene drives to control tick-borne diseases. Aided with understanding of scientific papers, CRISPR, and project brainstorming and planning. Received very positive feedback from the student's teacher as to the value of my help, and as such was requested to tutor another student in 2023 who's project will be about genetic engineering of bioluminescent worms for fishing bait. |
| Year(s) Of Engagement Activity | 2022 |