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INVESTIGATOR: EGNIN, M.

PERFORMING INSTITUTION:
TUSKEGEE UNIVERSITY
TUSKEGEE, ALABAMA 36088

EXPLORING NEXT GENERATION SWEETPOTATO BREEDING WITH CRISPR ASSOCIATED PROTEINS

NON-TECHNICAL SUMMARY: Genetic improvement of crops has become an issue of great interest in this era of big data agriculture and genome editing. The integrative nature of breeding and genome engineering offers new prospects for gene manipulation that translate molecular knowledge and principles to agriculture and food systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins systems (Cas) is a cutting edge technology utilized for gene editing. It has been used to edit the genomes of a number of organisms; thus, revolutionizing medical sciences in human cancer and Alzheimer research by creating precise mutations in mammalian cell genomes to generate beneficial genetic alterations. Genome editing technologies have been applied to numerous plants; thus, adding genes of value to crops. As the impacts of the genome editing revolution continue to gain roots across plant breeding research, scientists are poised to understand the functions of many more genes and to develop more efficient editing platforms. The ability to selectively alter genomic DNA sequences in organisms has opened new alternative avenues of research and breeding for difficult and orphan crops with multiple chromosome sets such as sweetpotato. Sweetpotato, known as yam in the US, ranks highest among all vegetables as a major source of calories and micronutrients, vitamins A and C, folate, iron, copper, calcium and fiber. It is grown by US farmers, ranks seventh in the world most important crops and is especially popular among limited resource farmers because of its adaptability, drought tolerance, and minimal or no chemical inputs requirement. The sweetpotato has evolved multiple strategies to cope with devastating stresses, however, the lack of true natural resistance to diseases or insect pests, compounded by low protein levels and essential amino acid profile, beset many cultivars. Sweetpotato has received limited research attention and its total potential has not been fully realized due to its complex genetic and vegetative propagation natures that restrict cultivar improvement in controlled plant breeding and genetic engineering. Thus, The opportunity to understand or accelerate sweetpotato improvement has immense potential in gene utilization and improvement of this crop. This project seeks to explore the feasibility of genome editing of sweetpotato, a six chromosome-set vegetatively propagated crop, through 4 objectives: 1) design and assemble gene editing constructs; 2) test the efficacy of these constructs through cell protoplast transfection; 3) perform stable Agrobacterium-mediated sweetpotato transformation, and; 4) detect and validate the resulting genetic changes, while training students and faculty. This exploratory research will use CRISPR technology with a single or two sgRNA constructs to target sites that can be edited simultaneously, causing mutation of the gene or deletion of the gene fragment between the two target sites. The design and assembly of this multiplex CRISPR/Cas9-sgRNA system will contribute not only to sweetpotato, but also to other root crop genetic improvement. These experiments will also test the efficiency of disrupting multiple gene copies in a multiple chromosome set species and verify the roles of molecular components involved in plant disease resistance and plant development. The information obtained from this study will potentially improve marker-assisted breeding protocols providing direct benefits for crop producers and stakeholders. The success of the project will help alleviate the breeding bottlenecks often present in underfunded and understudied staple crops. This research project is innovative in that it will develop the tools to conduct sweetpotato genome editing and, eventually, translate this knowledge into precision plant breeding for the development of sustainable systems for food and industrial feedstock production. This exploratory project on CRISPR/Cas-mediated sweetpotato genome editing represents a new strategy to improve sweetpotato, predictably, build the farming capacity, and bridge the technological gap most pronounced in the orphan crops. This will, in turn, accelerate breeding and many important endeavors in building 1890 Land Grant University research capacity. This project will be integrated into a breeding class to engage minority students in a setting where they can be creative in genome editing and subsequent phenotypic analysis; thus, contributing to create a network of sweetpotato plant resources to meet the nation's research and breeding requirements for the 21st century.

OBJECTIVES: The overarching goal of this exploratory project on developing sweetpotato genome editing using CRISPR/Cas is congruent with the strategic priorities of NIFA and the mission of Tuskegee University and other 1890 institutions in providing broader access to innovative technologies in support of standard-based research and learning. To investigate the feasibility of CRISPR-mediated mutagenesis of sweetpotato, we shall focus on hexaploid cultivated sweetpotato (PI318846 and TU-155) and Ipomoea trifida, the wild progenitor diploid species of cultivated sweetpotato. It is cross-compatibile with sweetpotato, shares numerous gene-clusters with potato, cassava, and Arabidopsis thaliana. Its smaller genome size and lack of chromosomal redundancy make it more amenable to genome editing than hexaploid sweetpotato. Four specific objectives will be carried out: 1) Design and assemble multiple CRISPR/Cas9-sgRNA-related vectors; 2) test constructs in sweetpotato protoplasts using T7 endonuclease assays; 3) perform stable Agrobacterium-mediated transformation and regeneration of sweetpotato; and 4) perform agronomic phenotyping and detection of mutations by DNA sequencing analysis of transgenics. The results of high editing efficiency to produce deletions, insertions, and substitutions will provide guidance to select efficient sgRNAs for sweetpotato hexaploid genome editing. This project draws on a new molecular technique for mutating genes, introducing key changes into genes (e.g. to make them easy to study in their native state), and mitigating regulatory issues of GMOs for the advancement of sweetpotato biotechnology and breeding sectors.