Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
(N/A)
Non Technical Summary
Biotechnology for crop genetic improvement becomes increasingly important for enhancing agricultural production. However, when introducing foreign genes into target crops using transgenic approach, or manipulating endogenous gene expression in target crops using genome editing for trait modification, the presence of undesirable DNAs permanently residing in the host genomes of the final transgenic products raises questions of potential hazards or adverse effects of these components to the host, environment and human health. it is therefore critical to remove these undesirable DNAs in the final products. Site-specific DNA recombinases catalyze reciprocal DNA exchange between specific DNA target sites and therefore can alter genomic DNA sequences in specific ways providing powerful tools in plant biotechnology and biosafety, for example their use for DNA excision in removing undesirable DNA to produce clean and environmentally friendly transgenics, and their use in developing gene containment strategies. However, the observations of recombinases-mediated unexpected phenotypic impact on model and crop plants raise valid concerns about the potentially unintended and off-target host phenotypic, genome and epigenome modifications by recombinases. The major objective of this project is to investigate how the commonly used site-specific recombinase genes, Cre, FLP and PhiC31, when constitutively expressed or induced in creeping bentgrass - an economically and environmentally important monocot perennial grass species, and in Arabidopsis - a dicot model plant, would have unintended off-target effects to host genomes, epigenomes and phenotypes, and thus negatively or positively affect plant traits including development, growth, and stress responses and present hazards to environment. We will examine whether and how the recombinases-mediated phenotypic changes are associated with unintended and off-target modifications in host gene expression and epigenetic modifications at global scale. Data obtained will allow feasibility evaluation of site-specific recombination system as a viable platform for gene excision to facilitate plant transformation and enable expanded application of genome editing and other biotechnology strategies for crop performance and biosafety enhancement. The research will provide information for regulatory agencies to assess site-specific recombination system-related biotechnology strategies developed for transgene excision and containment. The project specifically addresses program area 5a: "Research addressing phenotypic effects associated with unintended and off-target modifications in GE organisms developed using genome editing technology or other genetic engineering techniques and potential hazards or adverse effects (e.g., resistance) associated with these phenotypic effects to the environment". The project also indirectly addresses program area 1d, "Development or evaluation of effective strategies, including molecular and/or genetic, to limit gene transfer (gene flow) or outcrossing to sexually compatible organisms or transfer of genetic material between viruses, insects, or microorganisms".
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
The major objective of this project is to investigate how the commonly used site-specific recombinase genes, Cre, FLP and PhiC31, when constitutively expressed or induced in creeping bentgrass - an economically and environmentally important monocot perennial grass species, and in Arabidopsis - a dicot model plant, would have unintended off-target effects to host genomes, epigenomes and phenotypes, and thus negatively or positively affect plant traits including development, growth, and stress responses and present hazards to environment. We will examine whether and how the recombinases-mediated phenotypic changes are associated with unintended and off-target modifications in host gene expression and epigenetic modifications at global scale. There are five major objectives proposed in this project:Objective #1:Design and synthesize two groups of six Agrobacterium binary vectors for constitutive or inducible expression of three representative site-specific recombinase genes, Cre, FLP or PhiC31 (Year 1).The first group includes three constructs p35S/Cre-Ubi/hyg, p35S/FLP-Ubi/hyg, and p35S/PhiC31-Ubi/hyg for constitutive expression of Cre, FLP or PhiC31. The second group includes other three constructs pGmhsp17.5-E/Cre-Ubi/hyg, pGmhsp17.5-E/FLP-Ubi/hyg, and pGmhsp17.5-E/PhiC31-Ubi/hyg, each consisting of a soybean heat-shock gene promoter Gmhsp17.5-E driving either Cre, FLP or PhiC31 gene. All constructs have a corn Ubi promoter driving hygromycin resistance marker gene, hyg as a seleactble marker for plant transformation. In addition, a selectable marker gene hyg expression vector pUbi/hyg, and another construct for chemical-inducible expression of PhiC31, pPG10-90/XVE-OLexA-35/PhiC31-Ubi/hyg will be prepared as controls. There are three expression cassettes in the contruct for chemical-inducible expression of PhiC31: a G10-90 promoter drives the chimeric transcription activator XVE, a target promoter consisting of eight copies of the bacterial repressor LexA operator fused upstream of the -46 35S minimal promoter drives PhiC31, and a corn Ubi promoter drives hygromycin resistance marker gene, hyg as a seleactble marker for plant transformation. All the gene constructs will be prepared and validated using standard molecular biology techniques and DNA sequencing.Objective #2:Produce eight transgenic lines in both turfgrass and Arabidopsis that harbor the eight chimeric gene constructs prepared in Objective #1.(Years 1-2).Produce transgenic creeping bentgrass and Arabidopsis lines with the the aforementioned eight constructs.Objective #3:Evaluation of transgenic plants for off-target phenotypic changes including plant morphology and stress responses. (Years 2-3).To assess unintended off-target phenotypic effects by different recombinases in creeping bentgrass, we will choose three representative lines for each of the eight gene constructs along with transgenic controls without recombinase and investigate plant growth, development and plant performance under various abiotic stresses including drought, salt, heat, and nitrogen (N) starvation.Objective #4: Transcriptomic profiling by RNA-seq analysis to assess the potential recombinases-mediated off-target effects at genome-scale. (Years 3-4).To assess the risks of recombinases-mediated off-target genomic effects, we will conduct RNA-seq analysis for transcriptomic profiling of the transgenic creeping bentgrass and Arabidopsis lines with constitutive expressed or induced expression of recombinase genes in comparison with transgenic controls without recombinase.Objective #5: Genome-wide methylation analysis using bisulfite sequencing and transcriptome-wide m6A profiling using m6A-IP-Seq to reveal potential epigenomic impact of different recombinases expressed in host plants. (Years 3-4).Recombinase gene expression, whether induced or constitutive, might also cause unintended epigenomic changes to the host genome, and thereby affect gene expression in different biological pathways and result in off-target phenotypic effects, whether negatively or positively. We will conduct Genome-wide methylation analysis using bisulfite sequencing and transcriptome-wide m6A profiling using m6A-IP-Seq to reveal potential epigenomic impact of different recombinases expressed in host plants.
Project Methods
In this project, we will prepare a total of eight chimeric gene constructs, which will all be introduced into Agrobacterium tumefaciens by electroporation. The resulting strains will then be used for creeping bentgrass transformation via infection of embryogenic callus initiated from mature seeds and for Arabidopsis transformation using floral dip method to generate transgenic lines with constitutive expression or heat shock-induced expression of three representative site-specific recombinase genes, Cre, FLP or PhiC31. Transgenic plants will be examined for potential phenotypic changes including morphological characteristics, plant development and growth rate as well as plant responses to various abiotic stresses. RNA-seq will be conducted to exam the potential impact of the recombinases-mediated off-target effects on host gene expression. The recombinases-mediated potential epigenetic changes will also be examined by a genome-wide assessment of DNA and RNA methylation at global scale. Specifically, we will conduct genome-wide methylation analysis using bisulfite sequencing, and transcriptome-wide m6A profiling using m6A-IP-Seq to reveal potential epigenomic impact of different recombinases expressed in host plants.