Recipient Organization
UNIVERSITY OF WASHINGTON
4333 BROOKLYN AVE NE
SEATTLE,WA 98195
Performing Department
(N/A)
Non Technical Summary
Plant biotechnology is keyto developing foods, fuels, fabrics, and building materials that will help us adapt to and mitigate climate change. Plants can be engineered to resist drought and disease; to produce pharmaceuticals and other valuable bioproducts; and to replace industrial inputscurrently derived from petroleum.However, most genetically engineered plants currently suffer from a serious problem. Theversions of genes that we insert into plants often become "silenced"over time, so that they no longer produce anything useful. Moreover, it is very costly and time-consumingto "stack" multiple engineered traits into a single plant variety. If we could devise a way to transform plants with several genes at once, this would streamline the ability of American agriculturalcompanies to develop useful and profitable new crop varieties. Achieving that end is the focus of this research.The project seeks to discover genetic insulators, which are DNA sequences that lie in between genes to improve their behavior. Insulators have two main benefits. First, they ensure that genes are properly regulated, that is, turned on or off in the correct part(s) of the plant. Second, they serve as a genetic buffer,so that the genes produce a consistent productno matter where they are inserted in the genome. Wewill use next-generation DNA sequencing technology to screen many thousands of potential insulators. Using artificial intelligence techniques, we will build a computer model of insulation in plants. We will also test our designs in live plants over several generations to determine whether the insulator sequences perform as intended. If successful, this work will solve a longstanding issue in biotechnology, which in turn will benefit American companies, farmers, and consumers of staplecrops including corn, soy, and cotton. The project also funds hands-on training and mentorship forcollege students in the biological sciences, facilitating the development of a highly skilled agricultural workforce.
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
The goal of this research is to improve the properties oftransgenic plants used in agricultural biotechnology. Specifically, the project seeks to discover genetic insulators, which are DNA sequences that lie between genes to ensure that genes do not become silenced when inserted into plants. Insulators also regulate gene expression, ensuring that genes are only expressed in desired tissue types, rather than being expressed throughout the plant. In order to find and use novel plant insulator sequences, the following objectives will be pursued:Use a massively parallel reporter assay called plant STARR-seq to screen thousands of insulator candidatesUse machine learning algorithms and in silico evolution to strengthen the insulator candidates identified in the screenValidate insulator candidates for their effect on protein expression using a dual luciferase assayMake stable transgenic plant lines containing the insulator sequences discovered in steps 1-3Test a known insulator from animals in a plant modelUse novel insulator sequences to build a transgene cassette carrying multiple genes
Project Methods
Efforts (research): A massively parallel reporter assay developed byour lab, known as plant STARR-seq, will be used to screeninsulator candidates. A relative of tobacco called Nicotiana benthamiana will serve as amodel organism. The bacterial vector Agrobacterium tumefaciens will be used to transfect plasmidDNA libraries containing thousands of insulator candidates intoplant leaves. Two days later, RNA will be extracted from infected leaves andused to make sequencing libraries for the Illumina NextSeq platform. Sequencing data will be analyzed to determine the strength of insulator candidates in parallel. We will repeat the experiment using protoplasts madefrom maize leaves (Zea mays).A machine learning model for insulators will be built using the sequencing data. We will performin silicoevolution on the candidate sequences to attempt to produce even stronger insulators than are found in nature. We will also make stable transgenic plants in the model speciesArabidopsis thaliana. This will test whether promising insulator candidates actually work when integrated into the plant genome. Lastly, we will use the best insulators discovered in these experiments to build a multigene cassette. This will include three different fluorescent reporter genes that are regulateddifferently in order to show expression in specific plant tissues. These reporter genes will beflanked by insulator sequences that will hopefully ensure that the genes are only expressed in the target tissue types.Efforts (career, mentorship, and public outreach):The project director will mentor undergraduate students majoring in biology throughout the performance period. Undergraduate helpers will assist with data collection and plant care responsibilities. The project director will also help teach a summer course for high schoolers on basic computer programming and molecular biology techniques. The project director will participate inpublic science outreach events in the form of a lecture series. The project director will publish papers and attend large plant biology conferences to make connections in academia and in the biotech industry.Evaluation (research): Goals will be achieved if at least one novel genetic insulator is identified via the approach described above. Success in developing stable transgenic plants will be evaluated by achieving lowvariance in reporter gene expression among multiple lines of plants carrying the same DNA insert. Success in the insulation of the multigene cassette will be evaluated by directly viewing the tissue specificity of each reporter gene. Success in communicating research results will be met by publishing at least one peer-reviewed publication and attending at least one talk at a national/internationalconference.Evaluation (career, mentorship, and outreach):Success in translating the research to American agriculturewill be marked by meeting with at least one representative from a leading agriculture company to discuss the commercialization potential of the research. Career success will be achieved if the project director obtains an employment offer from a university or biotechnology company.Teaching and mentorship goals will be achieved if at least one undergraduate student graduates in biological science after working in the lab. Public outreach goals will be achieved if at least 50members of the public attend one or more of the lecture series at which I present.