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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.

APPROACH: To investigate the feasibility of CRISPR-mediated mutagenesis of sweetpotato, highly heterozygous/homozygous diploid I. trifida (wild progenitor) and hexaploid sweetpotato, obtained from the U.S. Sweetpotato Germplasm Repository, will be maintained in the greenhouse for subsequent analyses with seleced endogenous functional genes from our RNAseq analyses.(1) We shall first Design and assemble sgRNA expression cassettes containing the guide sequences easily integrated into sgRNA expression vectors using overlapping PCR chimeric primers. Protospacer adjacent motif sequences will be identified for each selected gene preceded by 19-25 base pairs. The sgRNA sequences will be compared to the sweeetpotato genome to assess the specificity of the designed. Next, sgRNA-coding sequences will be fused to the U6 promoter to U6/sgRNA cassettes then cloned into the donor vector pSAK2, using the flanking restriction endonuclease sites SwaI and SpeI. Followed by Cloning the U6/sgRNA expression cassette into a destination vector. For protoplast experiments, the U6/sgRNA expression cassette will be cloned into a pBlueScript-based plasmid containing a Cas9 gene under the control of a CaMV double 35S promoter. For stable transformation experiments, the U6-sgRNA cassette ligated into the final T-DNA binary vector p201N-Cas9 at the SwaI and SpeI sites. A minimum of 11 expression constructs with one or multiple sgRNA cassettes will be generated, and then used for sweetpotato protoplast transformation.(2) The sgRNA constructs will be tested using T7 endonuclease assays in protoplasts transiently to confirm that a particular sgRNA or sets are effective in generating mutations. Protoplasts will be generated from hexaploid sweetpotato and diploid I. trifida petiole and leaf explants. Fresh Protoplasts will be subjected to cell wall digestion using protoplast. Following digestion, protoplasts will be washed, resuspended and transfected with 4μg Plasmid DNA by PEG-mediated. After incubation for 20 min at room temperature, cells will be washed and collected by centrifugation at 200×g for 5 min. The cells will be resuspended, transferred to culture plates, and incubated at 25°C in the dark for 24 hours.Transfected protoplasts DNA will be tested for mutations using a T7 endonuclease I Surveyor assay. In this assay, the region of DNA to be mutated is amplified by PCR, denatured, then slowly renatured. Most of the amplicons will be wild-type sequence. They will form perfectly matched hybrids, which cannot be digested by T7 endonuclease I. However, amplicons derived from mutated sequences will form heteroduplexes with wild-type amplicons; the single-strand mismatch regions will be cleaved by T7 endonuclease I. Subsequent electrophoresis of the fragments through agarose gels will reveal mismatched regions as cleaved amplicons. The sizes of the cleaved amplicons would reveal where in the fragment the mutation has occurred. For situations in which multiple sgRNA constructs are used to generate deletions, PCR can be used directly to detect mutations. The extent of mutations will be estimated by scanning agarose gels and comparing the intensity of the wild-type amplicons with that of the mutant amplicons as cleaved bands for the T7 endonuclease assay, and smaller bands for two sgRNA-induced deletions. We shall use for stable transformation experiments with those sgRNA constructs that are most efficient in generating mutations.(3) Perform stable Agrobacterium-mediated transformation and regeneration of sweetpotato cultivars PI318846-3 and TU-155 and their wild diploid relative I. trifida. Several independent transgenic lines will be developed and genomic DNA will be extracted from leaves of transgenic regenerated plants. Genomic DNA will be amplified by PCR with primers flanking the predicted target sites, and the presence of mutations will be assessed using the T7 endonuclease I assay as described above. For those lines showing mutations, PCR products will be sequenced directly. Samples with heterozygous, biallelic, or (in the case of hexaploid sweetpotato) multiple allelic mutations will be decoded using the Degenerate Sequence Decoding method24. Moreover, we shall investigate whether the number of simultaneously expressed sgRNAs affects the editing efficiency significantly. Edited amplicons will be categorized to determine the types of editing (addition, deletion, or substitution) that have occurred using the various sgRNA constructs. We shall also assess the ability of the various constructs to generate homozygous, heterozygous, biallelic, or (in case of hexaploid cultivars) multiple allelic mutations in the diploid and hexaploid sweetpotato species. It may be easier to generate homozygous edited loci in diploid homozygous plants than in diploid heterozygous plants. Generating homozygous mutant alleles in a hexaploid may be still more difficult.One of the concerns of CRISPR/Cas9 mutagenesis is the generation of off-target mutations. To avoid or mitigate such mutations is to utilize existing algorithms and bioinformatics tools to design the sgRNA. Because the full genome Index of sweetpotato is not available, the specificity of the sgRNA will be checked based on the existing genome sequence of sweetpotato and comparing the designed sgRNAs with the genomic sequence of I. trifida to select specific sgRNAs. We shall perform deep sequencing of the mutants and compare these with wild-type genomic sequences to reveal the presence of off-target mutations.(4) As we utilize CRISPR-Cas9 genome editing to generate mutations, homozygous (or haplo-insufficient) mutants may exhibit a phenotype. For example, generation of homozygous phytoene desaturase (pds) mutants inhibits chlorophyll biosynthesis, resulting in "bleached" leaves. We shall first target sweetpotato PDS gene for mutagenesis because of the obvious bleached phenotype of the leaves of the regenerated plants. Additionally, targeted mutations will be tested by PCR-T7 endonuclease I assays on genomic DNA from protoplasts or transgenic leaves subjected to gene editing. Genetic sequencing analysis will be performed to identify loss or gain of nucleotides in the targeted genes and to identify sequence polymorphisms.Pitfalls: CRISPR/Cas9, is intrinsic to biology; however, its efficiency and specificity are affected by many features. A major issue for this project is the ploidy of the hexaploid sweetpotato, leading to the presence of six copies of each gene. Furthermore, sweetpotato is vegetatively propagated, making it difficult to generate homozygous mutants. CRISPR/Cas9 mutagenesis of all the copies of a given gene could be challenging, but very exciting as successful editing of multiple alleles has been achieved in hexaploid wheat. The wild diploid relative I. trifida will be used as an alternative first to develop the genome-editing tool of CRISPR/Cas9, and then improve mutagenesis of hexaploid sweetpotato.Year 1 will be dedicated to mining gene data for target sites, designing 4-8 CRISPR/Cas9-sgRNA-related vectors, assembling multiple sgRNAs, and conducting some activities for objectives 2 and 3. Objectives 3 and 4 will be fully carried out in Years 2 and 3. The results may suggest significant differences in the number of sgRNAs and the efficiency of the system. We foresee efficiency of our systems and we will plan ahead to complete the activities.Quarterly and yearly evaluations by both Dr. Jolly and the trainees will document the research progress and perceptions of the training, respectively. The information will help modify research and training, in case of pitfalls, for future activities. The evaluation will address these points: importance of the proposed activity to advancing knowledge and understanding for sweetpotato improvement; extent of the proposed activity resulting in creative, original, or potentially transformative concepts; and the broader impacts of the activity to expedite sweetpotato breeding.

PROGRESS: 2020/04 TO 2021/04
Target Audience:The target audience remains the same as stated in previous year to include scientific community, students, sweetpotato breeders and growers' associations, other root and tuber stakeholders, legume and vegetable research groups, Small and Big Data Farmers, and the general public interested in learning about the potential agricultural applications and benefit of this technology. Changes/Problems:The biggest challenge faced this reporting period was the COVID-19 pandemic and all the attendant issues including but not limited to restrictions on travel, gathering, workspace and significant delays in procuring supplies. While these impacted our workflow, creative solutions were quickly put in place to minimize impact on project outcome and deliverables. What opportunities for training and professional development has the project provided?This project provided resources for learning and sharing work outcomes, participation in professional meetings and conferences (National Association of Plant Breeding, NAPB; Society for InVitro Biology, SIVB; Professional Agricultural Worker Annual Conference; AAUW; FFAR) for students (A. Brown, F. Bukari, I. Ritte, and others) and faculty. Although in-person conferences and professional meetings were impacted by the global pandemic, students and faculty were still able to participate in different leaning and networking experiences virtually as well as telephone and email correspondences. The project helped to expose students to bioinformatics and other Big Data analytics and software training through a series of online workshops and webinars (DNASTAR, R, etc.). As part of outreach and bridge to STEM education for K12, we participated in Tuskegee University's summer AgDiscovery and AgriTrek Programs exposing 2 pupils directly to the CRISPR/Cas9 technology and indirectly to over 60 through their poster and oral presentations. In-class lectures through two courses (Plant Breeding and Scientific Communication) taught by the PI also provided an opportunity for introduction and training of more students (undergraduates and graduates) to the CRISPR/Cas9 technology and some of the techniques developed in this project. The project remains a great opportunity for collaborative exchanges between faculty at Tuskegee University (Drs. Egnin, Mortley and Bernard) and Purdue University (Drs. L-Y Lee and S. Gelvin). How have the results been disseminated to communities of interest?Results were disseminated through poster and oral presentations at scientific conferences (Society for InVitro Biology (SIVB); NAPB; Professional Agricultural Worker Annual Conference, CAST), virtual meetings on policy discussion with Congressional representatives at state and national level (Adrianne Brown), the outcomes from this project have also been presented by F. Bukari in partial fulfillment of her PhD Degree. Multiple manuscripts are in preparation for submission to scientific journals for peer review and publication. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete all activities under objective 4, make measurable progress on sustainability, and share the results with the scientific community and other stakeholders. We plan to complete the protein structure prediction, perform Cas9 RNP mediated protoplast transfection in sweetpotato varieties PI-318846, Beauregard and Resisto to test eIF4E knock-out function during SPFMV pathogen infection. We will prepare additional manuscripts for publication and improve the non-technical explanation of genome editing technology to impart to farmers and other stakeholders. Genes involved in Root-Knot Nematode and arsenic resistance have been identified in our lab and we are currently evaluating application of the lesson learnt in this project for elucidating mechanisms of resistance and tolerance in other polyploids and for future editing toward crop improvement.

IMPACT: 2020/04 TO 2021/04
What was accomplished under these goals? n this reporting period, we built on work done on objectives 1, 2, and 3 in the last reporting period and finalized objective 4 on the a) PDS work and b) the eIF4E isoform characterization. Under the PDS work, protoplast isolation and PEG-calcium transfection protocols were optimized and generated reproducible yield of 2.67 x 106 protoplasts/ml with 94.7% viability and 92% expression efficiency confirmed by YFPs localizations analysis. A novel nursed-protoplast culture regeneration protocol developed as part of the protoplast system produced embryos in less than 8 weeks and plantlets as early as 8 to 10 weeks. The Ipomoea batatas phytoene desaturase (ibpds) gene was cloned and characterized, and analysis revealed 2 to 4 allelic forms of ibPDS from sweetpotato cultivars N.Z. 196, Jewel, Nugget, Beauregard and Resisto, and DMOI, TUO2, and TU-purple (Tuskegee University breeding lines), suggesting ibpds is likely a member of multigene families, and the differences in ibPDS haplotype variants likely play a role in the differences in storage root color and carotenoid contents. Objective 4 was finalized with the selection of the conserved regions within exons 2, 5, 6, and 7 ibpds gene targeted by CRISPR/Cas9 editing. This resulted in regenerated edited plantlets expressing albino and dwarf phenotypes with induced mutations rates of 11-20% within the four target regions of the ibpds gene. These editing events were confirmed by PCR-based T7-Endonuclease-I assays, phenotyping, and sequence analysis. Under b) following the successful annotation of eIF4e within the four hexaploid sweetpotato through direct PCR sequence, predicted protein sequences conserved for eIF4e domains were generated and utilized for phylogenetic analysis with eIF4E sequences collected from NCBI Genbank of other species. Direct PCR sequence analysis revealed three non-synonymous mutations in Resisto eIF(iso)4E and non-synonymous mutations in Jewel eIF4E. The DNASTAR NovaFold protein modeling application was utilized to compare the predicted protein structural variation amongst the non-synonymous mutations (analyzed from the sequenced annotation results) to understand the observed differences within each variety. A total of 13 single guide RNAs (sgRNA) was designed for the CRISPR/Cas9 mediated site-directed mutagenesis. This will enable the targeting of conserved regions of each gene within the first three exon regions to initiate gene inactivation (eIF4E-4, eIF(iso)4E-5, CBP-4). An in-vitro cleavage activity assay was performed to screen the efficiency of each gRNA and out of the 13 sgRNAs evaluated, 5 gRNAs showed efficient cleaving activity. These 5 efficient gRNAs will be utilized for Cas9 RNP mediated protoplast transfection in sweetpotato varieties PI-318846, Beauregard and Resisto.

PUBLICATIONS (not previously reported): 2020/04 TO 2021/04
Type: Other Status: Published Year Published: 2021 Citation: Bukari, F. 2021. Development of an Efficient System for Characterization of Phytoene Desaturase Gene and CRISPR-Cas9 Mediated Gene Editing in Hexaploid Sweetpotato. PhD Dissertation Tuskegee University. Major Professors: Marceline Egnin and Deloris Alexander Idehen, O., M. Egnin, R. Ankumah, R. Shange. 2021. Evaluation of phenotypic and biochemical responses of Pteris vittata during growth in arsenic contaminated soil and its effect on selected soil enzymes activity. In Press: African J. of Agriculture Research (AJAR/16.12.20/15410). Bukari, F., M. Egnin, O. Idehen, G.C. Bernard, A. Brown, D. Mortley, C. Bonsi, D. Alexander, L-Y. Lee and S. Gelvin. 2021. Development of an Efficient System for Characterization of Phytoene Desaturase Gene and CRISPR-Cas9 Mediated Gene Editing in Hexaploid Sweetpotato (Manuscript in preparation (Invitro Developmental biology-Plant to be submitted).

PROGRESS: 2017/04/15 TO 2018/04/14
Target Audience:Our target audience will be beyond the expected scientific community to include students and stakeholders, sweetpotato breeders and grower associations, other root and tuber, legume and vegetable researchers, big data farmers, and the general public interested in learning the technology. Changes/Problems:No major change except for expanding the cultivar base to 12 in order to realize the full potential the heterogeneous nature What opportunities for training and professional development has the project provided?The project was a great opportunity for students, staffs and faculty at Tuskegee and Purdue Universities to learn the different steps involved in CRISPR/Cas9 mediated genome editing technology as collaborative exchanges between the two universities. The application of the CRISPR/Cas9 tool requires different skills of molecular biology such as the design of the gRNA (single or multiple), assembling of the cassettes, the cloning of the cassette into different expression vectors as well as the transformation or trans-infection technology. The project provided students and faculty the opportunities to learn about the CRISPR/Cas9 genome editing system as well as other existing gene editing tools. At faculty level, gene editing classes were developed at Tuskegee University in the iBREED program as well as at the graduate level in the Department. Dr Traore have thought several classes related to the technology as well as it impacts in the sweetpotato breeding. PhD students Foazi Bukari and Adrianne Brown have gained trmendous knowledge of this technolofy on this project. For sweetpotato breeders and farmers, several activities have involved the explanation of the genome editing technology, specifically the CRISPR/Cas9 mediated genome editing. Several students and faculty have visited the Gelvin's Lab at Purdue University for technology and knowledge transfer working with Dr. Lee. How have the results been disseminated to communities of interest?The results have been disseminated through scientific conferences and meetings. Our group has participated in the ARD meeting (2017) for oral presentation and InVito Biology (2017) for poster presentations. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete work on objective 2, start objectives 3 and 4, and share the results with the scientific community and other Stakeholders. The cloned and sequenced pds genes will be characterized and utilized to design and assemble several gRNA cassettes, which will then be cloned into pUC119 for protoplast transfection and p201N/Cas9 for plant tissue transformation. Other genes related to nematode resistance, storage root development and those indicated in the project ,will be amplified and characterized for subsequent guide constructs for gene editing.

IMPACT: 2017/04/15 TO 2018/04/14
What was accomplished under these goals? To test whether CRISPR/Cas9 can be used as a technology to edit the sweetpotato hexaploid genome as a proof of concept, we developed and optimized a repeatable protoplast system from an existing one for transfection and cell regeneration. Our results exhibited high quality and yield levels of intact spherical sweetpotato protoplasts devoid of contaminant and infection during culture. To test sweetpotato protoplast viability and its ability to express genes, sweetpotato protoplasts were trans-infected with the venus gene coding for a fluorescence protein fused to different cell localization signal peptide as a visual marker. Our results have demonstrated that sweetpotato protoplasts could be utilized as a system for gene expression in different compartments of the cell, including the nucleus, cytoplasm and the plasma membrane. Phytoene desaturase gene is involved in chlorophyll biosynthesis/carotenoid content, in sweetpotato, storage root skin and flesh varied in color from white to deep purple based on carotenoid contents, and since pds gene is a member of multiple gene families, carotenoid contents may be intrinsic to differences in copy numbers critical to storage root flesh/skin color variation from white to dark- purple, The goal of testing the efficiency of CRISPR/Cas9 in sweetpotato protoplast as a proof of concept was accomplished with Arabidopsis phytoene desaturase (pds) gene by silencing the homologous pds gene in sweetpotato, which will impair the formation of pigment in leaf as well as storage roots, resulting in a bleaching phenotype of the leaves. Following co-transfection with PUC119/gRNA and HBTpco/Cas9 and 48hours co-cultures, genomic DNA were extracted followed by PCR and T7 endonucleases assays resulting in successful editing in Trifida and sweetpotato protoplasts. However, due to hexaploid nature we uncovered other polymorphisms in the differing cultivars. Thus, the pds gene from sweetpotato cultivars with differing storage root, skin and flesh color (from white to deep purple) were amplified from genomic DNA, cloned and sequenced.

PUBLICATIONS: 2017/04/15 TO 2018/04/14
Type: Book Chapters Status: Published Year Published: 2017 Citation: Bernard, G., M. Egnin, C. Bonsi. 2017. The Impact of Plant-Parasitic Nematodes on Agriculture and Methods of Control. In Press: InTech "Nematology", ISBN978-953-51-55270 http://creativecommons.org/ BUKARI, F., S. Traore, M. Egnin, C. Bonsi, S. Gelvin, G. Bernard, O. Idehen. 2017 CRISPR/Cas9-sgRNA mediated multiplex gene modification in Sweetpotato. In-Vitro Dev Plant, vol 53 (A):12.