Progress 09/01/13 to 08/31/18
Outputs
Target Audience:Our research effort has been made to outreach to and benefit a wide range of audience with our findings that reflect the advance of genome editing technology development and application in plants, especially in agriculture. The audience includes general public, e.g., growers and extension specialists, scientists from university and industries, university and government administrators. We communicate with the audience by publishing our findings (research results) in scientific journals, giving oral presentation in national and international conference/meetings, seminars and even college courses. Graduate students and post-doc benefit by participating in this research project as their major training programs. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project of the last year has provided training opportunities for one graduate student and one scientist to work on the plant physiology, morphology and biology. How have the results been disseminated to communities of interest?In the last year of project, the results have been disseminated among scientific community, university and industry through publication of peer-reviewed article and oral presentations. we published one peer-reviewed article, gave multiple presentations related to the project in universities, workshops and conferences with the attendance of scientists, students, industry and regulatory people. For example, PIs have presented the work and acknowledged the NIFA funding in the following conferences as the invited speakers: 1. The 6th Xanthomonas Genomics Conference, Halle, Germany, July 20, 2018 2. The Plant CRISPR Workshop: CRISPR/Cas-mediated Plant Genome Editing: From Design to Analysis, Nairobi, Kenya, June 19-20, 2018 3. DuPont/Pioneer, Johnston, Iowa, May 25, 2018 4. The 11th Canadian Plant Biotechnology Conference, Saskatoon, Saskatchewan, Canada, May 16, 2018 5. Department of Plant Science & Landscape Architecture, University of Maryland, College Park, Maryland, March 8, 2018 6. The 54th Annual Illinois Corn Breeders' School, Champaign, Illinois, March 5, 2018 7. Division of Plant Sciences, University of Missouri, Columbia, Missouri, January 18, 2018 8. CSIRO Gene Editing of Crop Workshop, Kiama, NSW Australia, November 29, 2017 9. The 5th Plant Genomics & Gene Editing Congress: USA, Philadelphia, Pennsylvania, November 2-3, 2017 10. Department of Plant Pathology, University of Florida, Gainesville, Florida, October 24, 2017 11. Switchgrass Conference IV, Lincoln, NE, August 5, 2017 (presented by Shui-Zhang Fei, co-PI) What do you plan to do during the next reporting period to accomplish the goals?Overall Impact A. Scientific problem/issue and project relevance to agriculture The project was designed and performed to address the risk associated with TALEN-based genome editing in crop plants. The risk that was unknown a few years ago is directly related to an agricultural problem. First, TALENs or other genome editing technologies in general are developed to make precise, efficient and targeted genomic modifications in crops. However, it is unknown if TALENs or others would cause unintended off-target mutations in the genomes of interest, and if any, to what extent the off-targeting occurs. Second, when TALENs or other mutagens are introduced as DNA into plant cells, we expect the transgenes would be segregated out through genetic crossing in progeny plants. Therefore, the progeny containing the desired genomic modifications but lacking the transgene (so-called null segregants) will be selected, resulting in novel germplasm. It is also true that to what extent the risk of the transgene residual exists in the edited crop genomes. B. Major activities and outcome of project We have optimized a pipeline and protocol to design and engineer TALEN (TAL effector nuclease) constructs for genome editing in plants. The system is robust in rice, but inefficient in switchgrass as tested. Beyond the proposed scope, we developed two robust CRISPR/Cas9 based genome editing systems, one for rice and another for maize (Obj. 1). By applying next generation sequencing technologies (Illumina, PacBio) to whole-genome sequence 7 mutants and their parental line Kitaake that underwent two rounds of TALEN-mediated genome editing in the promoters of two SWEET (sugar transporter) genes, we found that thousands of variations (single nucleotide variants, insertions and deletions) between individual mutant lines and the wild type parent line. It is very hard to interpret the data to conclude those variations are due to off-target of TALENs or mutations induced by two rounds of tissue culture and transformation with TALENs. Alternative approach would be to whole-genome sequence multiple control lines that undergo the same transformation process with construct lacking the TALEN transgene, and tease out the transformation related DNA changes (Obj. 2). However, the whole-genome sequencing data told us that some (5 out 7) mutant lines became transgene-free, leading to null segregants that could be ruled as non-regulated articles by the the USDA APHIS BRS (Obj. 3). Finally, we have obtained new germplasms that contain the broad-spectrum disease resistance but show no obvious negative effect on other aspects of plant growth, deveolpment and reproduction (Obj. 4). While all those were unknown when the awarded project started. C. Implication of findings Our results demonstrate that TALENs as one type of engineered nucleases represent a highly specific and efficient tool for crop improvement with the potential to rapidly and precisely generate useful and novel genetic variations/traits. TALENs in particular, or engineered nucleases in general, as the mutagen for targeted genetic changes in plant cells and, after completion of their task in one generation, can be sorted out through genetic crossing and segregation in subsequent generations, resulting in transgene-free crop plants. The process seems different from the natural means or conventional mutagenesis, but the transgene-free products are not different. The TALEN-based product, if no transgenic DNA retained in the targeted genome, does not readily fit current definitions of genetically engineered or genetically modified organism (GMO) used within most regulatory regimes. Regulatory focus needs to be on the nature of the novel phenotype or product developed rather than on the process. The TALEN system we tailored for mutagenesis in switchgrass, unfortunately, did not yielded any detectable mutations as tested with three switchgrass genes, indicating the possible inefficiency of TALENs in this crop likely due to its complexed genomes (tetraploid of large genomes). The complexed and big genomes of crops in general have issue of gene silencing and efficiency for the nucleases to locate the right targets to induce double stranded DNA breakages, thus leading to much lower frequency of mutagenesis. On the other hand, lower on-target efficiency of genome editing means less chance of off-targeting in targeted plants, a positive prospect that the regulatory agencies may look out for. D. Impact of project The completion of this project has significant impacts on scientists, producers, regulatory agencies and even general public. The genetic engineering or gene editing in crop has been a hot topic debated by a wide range of audience for the last decades. The cost, length and uncertain outcoomes of the regulatory process prevent public sectors and small innovative entrepreneurs from pursuing GE crop research and production. An exemption or a less expensive and complex regulatory process would stimulate innovative research and development in GE technology to improve food security and environmental quality, which would depend on the research data from the risk assessment associated with specific technology. E. Relevance to federal regulatory agencies The completion of the stated objectives helps develop a better assessment of the TALEN-associated risks, provide a broad foundation for future crop engineering efforts directed at genomic modification in rice or other crop plants, provide comprehensive insight into the applicability of TALEN or other genome editing technologies to other crop and bioenergy plants, and assist federal regulatory agencies in determining whether TALEN technology merits either an exemption from regulatory oversight or a less rigorous regulatory process. F. Outcome metrics Total number of peer-reviewed publications 1. Yang Liu et al. 2018. Plant Biotechnology Journal 16(2):381-393. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5787850 2. Si Nian Char, et al. 2017. Plant Biotechnology Journal 15(2):257-268. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5259581/pdf/PBI-15-257.pdf 3. Ting Li, et al. 2016. Journal of Genetics and Genomics 43(5): 297-305. https://www.ncbi.nlm.nih.gov/pubmed/27180265 4. Donald Weeks, et al. 2016. Plant Biotechnology Journal 14(2): 483-495. https://onlinelibrary.wiley.com/doi/epdf/10.1111/pbi.12448 5. Jeffrey Wolt et al. 2016. Plant Biotechnology Journal 14(2): 510-518. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5042095/pdf/PBI-14-510.pdf 6. Si Nian Char, et al. 2015. Plant Biotechnology Journal 13(7):1002-1010. https://onlinelibrary.wiley.com/doi/abs/10.1111/pbi.12382 Total number of students trained: 2 Scientist and postdoc, 3 graduate students, and 2 undergraduate students. Total number of presentations: Forty one invited presentations related to and acknowledged the project have been given in various conferences and universities. The presentations include 17 in the international settings (Germany, United Kingdom, Japan, S. Korea, Thailand, Philippines, Taiwan, China, Australia, Brazil, Colombia, Canada) and 24 in US. Total number of patents filed or awarded: 1 patent awarded (Bing Yang, Ting Li and Bo Liu, Genetically modified plants with resistance to Xanthomonas and other bacterial plant pathogens. US9688997B2).
Impacts
What was accomplished under these goals?
Objective 1. Establish a pipeline of TALEN-based gene editing and assess its robustness and general applicability in crop plants by focusing on rice and switchgrass (Year 1-3 of the project) - 100% completion. Objective 2. Assess potential off-target mutations caused by TALENs in modified rice genomes by comparing seven modified genomes against the parental reference genome (Year 2-4 of the project) - 100% completion. Objective 3. Assess the removal of the TALEN transgenes from the modified rice genome through genetic segregation at the genomic level(Year 2-3 of the project) - 100% completion. Objective 4. Analyze potential phenotypic variations in rice plants associated with TALEN-based gene editing by investigating a number of morphological traits (plant height, tiller number, dry biomass, etc.) (Year 3-5 of the project) - 100% completion. The outcome of the results in the last year (no-cost-extension): We carried out the final field test in this summer (no-cost-extension) and did the data analysis based on the experimental data of three years. The results are consistent with the previous results. Specifically, we conducted small-scale field test in the ISU research farm with four rice lines that had been ruled by the USDA APHIS BRS as non-regulated articles. By analyzing the multiple-year data, we did not find obvious negative effect of the SWEET (sugar transporter) gene promoter mutations on the overall plant growth and production of those mutant lines relative to the parent cultivar Kitaake. For example, the overall hights (cm) range from 61.2±2.8 to 65.5±4.1 in 4 mutant lines vs 61.2±4.8 of the parent Kitaake; tiller number range from 6.8±1.1 to 7.1±1.3 in mutants vs 8.0±2.5 in Kitaake; flag leaf lengths range from 14.7±0.9 to 17.5±0.6 in mutants vs 16.5±0.4 in Kitaake; and seed setting (filled seeds/filled+empty seeds) range from 0.61±0.18 to 0.74±0.1 in mutants vs 0.77±0.12 in Kitaake.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Yang Liu, Paul Merrick, Zhengzhi Zhang, Chonghui Ji, Bing Yang and Shui-zhang Fei. 2018. Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9. Plant Biotechnology Journal. 16 (2): 381-393. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5787850/
|
Progress 09/01/16 to 08/31/17
Outputs
Target Audience:Our research effort has been made to outreach to a wide range of audience with our findings that reflect the advance of genome editing technology development and application, especially in agriculture. The audience includes, but not limited to, general public, graduate students, undergraduate students,scientists from universitiesand industries, and university and government administrators. We communicate with the audience by publishing our findings (research results) in scientific journals, giving oral presentation in conference/meetings, seminars and even college courses. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This grant has provided training opportunities for two graduate students and one undergraduate student as well as one scientist to work on the most advanced biotechnologies that provides promise in basic and applied science in agriculture. How have the results been disseminated to communities of interest?We have published multiple articles related to this project and supported by the award. The publication is one of the most efficient ways to disseminate our results among the scientific community. The PIs have also presented our effort and results in many scientific conferences and university lectures. For example, PI has presented the work and acknowledged the NIFA funding in the following conferences as the invited speaker: 1. The VI Brazilian Symposium of Plant Molecular Genetics, Ouro Preto, Brazil, May 29, 2017 2. Plant Pathology Department, Kansas State University, Manhattan, KS, May 4, 2017 3. Plant and Animal Genomics (PAG), Development and Application of Transgenic Technology in Agriculture Workshop, San Diego, CA, January 15, 2017 4. Plant and Animal Genomics (PAG), Plant Genome Engineering Workshop, San Diego, CA, January 15, 2017 5. TALEN- and CRISPR/Cas9-based gene editing in maize and rice. Cold Spring Harbor Laboratory Asia Conference, Awaji, Japan, November 30, 2016 6. Tokyo University of Science, Tokyo, Japan. November 24, 2016 7. Institute of Plant and Microbial Biology, Academia Sinica, Taiwan, October 26, 2016 8. National Institute of Agricultural Sciences, Rural Development Administration, R. of Korea, October 24, 2016 9. The 5th International Congress of Bacterial Blight of Rice, Manila, Philippines, October 18, 2016 10. 8th Annual Donald Danforth Plant Science Center Fall Symposium, St. Louis, MO, September 30, 2016 11. QTL Engineering Workshop, University of Minnesota, Twin Cities, MN, September 18, 2016 12. Life Science Seminar series, South Dakoda State University, Brookings, SD, Nov. 2015 13. The joint annual meeting of The American Society of Agronomy, Crip Science Society of America, and Soil Science Society of America, Symposium - Gene Editing for Crop Improvement, Minnesota, Nov. 2015 14. Physiological and Molecular Plant Biology Seminar series, University of Illinois, Urbana-Champaign, IL, Oct., 2015 15. CRISPR-Cas Workshop. John Innes Center, Norwich, UK, Sept. 2015 16. The 4thInternational Conference on Biotic Plant Interactions, Nanjing, China, Aug. 2015 17. The 5thXanthomonas Genomics Conference, Bogota, Colombia, June, 2015 18. CROPS 2015: Translating genomic knowledge into plant breeding and crop improvement, Huntsville, AL, May 2015 19. The 2015 In Vitro Biology, Tucson, AZ, May-June, 2015 20. Analytical Excellence through Industrial Collaboration hosted by Illumina, San Diego, CA, April 2015 21. The 57thAnnual Maize Genetics Conference, St. Charles, Illinois, 2015 22. The 12thInternational Symposium of Rice Functional Genomics, Tucson, Arizona, Nov., 2014 23. North American Agricultural Biotechnology Council 26, Ithaca, New York, Oct., 2014 24. The 4thInternational Rice Congress, Bangkok, Thailand, Oct., 2014 25. The 13thInternational Conference on Plant Pathogenic Bacteria, Shanghai, June, 2014 26. Snips & Pics: From Genome Targeting to Imaging, a short course hosted by the Genes & Development Graduate Program at the University of Texas MD Anderson Cancer Center, Houston, January 29-30, 2014 What do you plan to do during the next reporting period to accomplish the goals?We will request for a no-cost extensionto continue to work on Objective 4with the field test this summer, including wrapping up the ongoing field trial by 8/31/2017 and completing the data analysis, disseminate the results in publication by 1/31/2018.
Impacts
What was accomplished under these goals?
Objective 1. Establish a pipeline of TALEN-based gene editing and assess its robustness and general applicability in crop plants by focusing on rice and switchgrass. We have completed 100% (basic) of the proposed aim. We have tested and optimized a pipeline of designing and engineering TALEN (transcription activator-like effector nuclease) gene constructs based on the "Gold Gate" modular assembly method in rice. We used the rice system to make many TALEN constructs including 7 targeting the coding sequences of 7 rice SWEET (sugar efflix transporter) genes for knockouts and 3 targeting the promoters of two SWEET genes. Knockout mutants were characterized in detail for nature of mutations, their roles in rice plant growth, development and production. One of SWEET gene (SWEET4) has been found to be required for rice endosperm filling (Sosso et al., 2016 Nature Genetics 47, 1489-1493). The SWEET promoter mutants were characterized for bacterial blight resistance. Mutations in SWEET11, 13 and 14 enabled plants resistance to bacterial blight of rice (Zhou et al. Plant Journal 82, 632-643; unpublished data). We continued to characterize the seven TALEN rice lines with SWEET genes mutated including off-targeting by the engineered TALENs. Targeted PCR-amplification of relevant regions spanning the predicted off-target sites in three SWEET genes (SWEET11, 13, and 14) and Sanger sequencing of those amplicons yielded no detectable off-target mutations. We have also improved the protocol for switchgrass tissue culture and Agrobacterium-mediated transformation which is routine now in the co-PI, Dr. Shuizhang Fei's lab. We could not detect mutations from the switchgrass transgenic plants with TALEN constructs targeting three switchgrass genes. The data indicates the possible inefficacy of TALEN-based mutagenesis in switchgrass due to its complexgenome (tetraploid and big genome). Meanwhile, we expanded our effort to establish a CRISPR/Cas9 system in switchgrass. An efficient switchgrass CRISPR system has been established. We have successfully used it to mutate three switchgrass genes at high-efficiency (11-95%) in stable transgenic switchgrass plants (Liu et al. 2017 Plant Biotechnology Journal in press). We expanded our research effort to develop another genome editing technology, CRISPR based genome editing in rice and maize (Zhou et al. 2014 Nucleic Acids Research 42 , 10903-10914; Char et al. 2017 Plant Biotechnology Journal 15(2):257-268). Our rice and maize CRISPR systems have been widely shared among the scientific community worldwide. Objective 2. Assess potential off-target mutations caused by TALENs in modified rice genomes by comparing seven modified genomes against the parental reference genome. We have completed 100% (basic)of the proposed aim. We have whole-genome-resequenced 8 rice lines including one parental line Kitaake and seven lines that contained the TALEN-mediated edit in the promoters of SWEET11 and SWEET14 using the next generation sequencing technologies: Illumina and PacBio technologies. We have collaborated with Dr. Volker Brendel's group at University of Indiana to assemble the genomes and to perform comparative analysis. The results demonstrate that there are so many (thousands) of polymorphisms (SNPs, indels) among individual mutant lines and between them and the wild type parent line. It is very extremely hard to interpret the data to conclude those variations are due to off-target of TALENs. Instead, it is most likely that those deteded variations were caused by the processes of two rounds of tissue culture and transformation with TALENs. The conclusion from our work is that it is impossible to use whole-genome-resequencing approach to assess the genome wide off-target effect by TALENs if tissue culture and transformation process are involved. Objective 3. Assess the removal of the TALEN transgenes from the modified rice genome through genetic segregation at the genomic level. We have completed 100% (basic)of the proposed aim. The analysis of seven genomes that underwent two rounds of TALEN modification reveals that five out of seven segregants lines do not contain the DNA sequence of the TALEN constructs (transgene of TALENs and hygromycin resistant gene of transformation marker) at a 19 bp cut-off while the remaining two lines contain the residual transgenes. The results have validated our initial hypothesis that TALENs enable precise genetic editing and can be later segregated out through genetic crossing, leading to production of null segregants free of transgenes. The five rice lines have been ruled as non-regulated articles by the USDA APHIS BRS. This was completed in 2015. Objective 4. Analyze potential phenotypic variations in rice plants associated with TALEN-based gene editing by investigating a number of morphological traits (plant height, tiller number, dry biomass, etc.). We have completed 85% (basic)of the proposed aim and request for a no-cost extension of 5 months. We have conducted a small-scale field test in the ISU research farm with the five rice lines that had been ruled by the USDA APHIS BRS as non-regulated articles. The field test was performed in the summer of 2015 and 2016. From the last two summer tests, we did not find obvious negative effect of the SWEET gene promoter mutations on the overall plant growth and production of those mutant lines relative to the parent cultivar. We are planning to carry out the final field test in this summer and will do the data analysis based on the three-years experimental data. Impactful aspects of the results: Our results demonstrate that TALENs as one type of engineered nucleases represent a highly specific and efficient tool for crop improvement with the potential to rapidly and precisely generate useful novel genetic variations/traits. TALENs in particular, or engineered nucleases in general, as the mutagen for targeted genetic changes in plant cells and after completion of their task in one generation, can be sorted out through genetic cross and segregation in subsequent generations, resulting in transgene-free crop plants. The process seems to be different from the natural means or conventional mutagenesis, but the transgene-free products are not different. The TALEN-based product, if no transgenic DNA retained in the targeted genome, does not readily fit current definitions of genetically engineered or genetically modified organism (GMO) used within most regulatory regimes. Regulatory focus needs to be on the nature of the novel phenotype or product developed rather than on the process.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2017
Citation:
Yang Liu, Paul Merrick, Zhengzhi Zhang, Chonghui Ji, Bing Yang, and Shui-zhang Fei. 2017. Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L.) using CRISPR/Cas9. Plant Biotechnology Journal. In press.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Si Nian Char, Anjanasree K. Neelakandan, Hartinio Nahampun, Bronwyn Frame, Marcy Main, Martin H. Spalding, Philip W. Becraft, Blake C. Meyers, Virginia Walbot, Kan Wang, Bing Yang. 2017. An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize. Plant Biotechnology Journal 15(2):257-268. DOI: 10.1111/pbi.12611
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Ting Li, Bo Liu, Chih Ying Chen, Bing Yang. 2016. TALEN-mediated homologous recombination produces site-directed DNA base change and herbicide-resistant rice. Journal of Genetics and Genomics 43(5): 297-305. https://doi.org/10.1016/j.jgg.2016.03.005
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Donald P. Weeks, Martin H. Spalding and Bing Yang. 2016. Use of designer nucleases for targeted gene and genome editing in plants. Plant Biotechnology Journal 14, 483495. DOI: 10.1111/pbi.12448
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Jeffrey D. Wolt1, Kan Wang and Bing Yang. 2016. The Regulatory Status of Genome-edited Crops. Plant Biotechnology Journal 14, 510-518. DOI: 10.1111/pbi.12444
|
Progress 09/01/15 to 08/31/16
Outputs
Target Audience:We made our efforts to distribute our data and results to a diverse audience in terms of publication in scientific journals and oral presentation in conference/meetings and seminars. The audiences include general public, graduate and undergraduate students, scientists from university and industries, and university and government administrators. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This grant has provide trainning opportunities for two graduate students and one undergraduate student as well as one scientist to work on the most advance biotechologies that provids promise in basic and applied science in agriculture. How have the results been disseminated to communities of interest?We have published one article related to this project. The publication is one of the most efficient ways to disseminate our results among the scientific community. The PIs have also presented our effort and results in several scientific conferences and university lectures. For example, PI has presented the work and acknowledged the NIFA funding in the following conferences as the invited speaker: Life Science Seminar series, South Dakoda State University, Brookings, SD, Nov. 2015 The joint annual meeting of The American Society of Agronomy, Crip Science Society of America, and Soil Science Society of America, Symposium - Gene Editing for Crop Improvement, Minnesota, Nov. 2015 What do you plan to do during the next reporting period to accomplish the goals?We will continue to work on the objectives including wrapping up the ongoing field trial and disseminate the results in publication.
Impacts
What was accomplished under these goals?
Objective 1. Establish a pipeline of TALEN-based gene editing and assess its robustness and general applicability in crop plants by focusing on rice and switchgrass. 1.1. We have tested and optimized a pipeline of TALEN design and engineering based on the "Gold Gate" modular assembly method in rice. We continued to characterize the seven TALEN rice lines with SWEET genes mutated including off-targeting by the engineered TALENs. Detailed characterization of those seven SWEET genes revealed the important role of OsSWEET4 in rice seed filling (Sosso et al. 2015 Nature Genetics 47,1489-1493). 1.2. We have also improved the protocol for switchgrass tissue culture and Agrobacterium-mediated transformation which is routine now in the co-PI, Dr. Shuizhang Fei's lab. We could not detect mutations from the switchgrass trangenic plants with TALEN constructs targeting three switchgrass genes. The data indicates the possible inefficacy of TALEN-based mutagenesis in switchgrass due to its complexed genome (tetraploid and big genome). Meanwhile, we expanded our effort to establish a CRISPR/Cas9 system in switchgrass; we are currently characterizing the CRISPR plants. 1.3. We expanded our research effort to develop another genome editing technology, CRISPR based genome editing in rice and maize. Our CRISPR systems have been widely shared among the scientific community. We have completed 90% of the proposed aim. Objective 2. Assess potential off-target mutations caused by TALENs in modified rice genomes by comparing seven modified genomes against the parental reference genome. We have whole-genome-resequenced 8 rice lines including one parental line Kitaake and seven lines that contained the TALEN-mediated edit in the promoters of OsSWEET11 and OsSWEET14. The sequencing work with Illumina and PacBio technologies has been completed. We are currently collaborating with Dr. Volker Brendel's group at University of Indiana to assemble the genomes and to perform comparative analysis. We have completed 70% of the proposed aim. Objective 3. Assess the removal of the TALEN transg enes from the modified rice genome through genetic segregation at the genomic level. The analysis of seven genomes that underwent two rounds of TALEN modification reveals that five out of seven segregant lines do not contain the DNA sequence of the TALEN constructs (transgene of TALENs and hygromycin resistant gene of transformation marker) at a 19 bp cut-off while the remaining two lines contain the residual transgenes. The results have validated our initial hypothesis that TALENs enable precise genetic editing and can be later segregated out through genetic crossing, leading to production of null segregants free of transgenes. The five rice lines have been ruled as non-regulated articles by the USDA APHIS BRS. This was completed in 2015. We have completed 100% of the proposed aim. Objective 4. Analyze potential phenotypic variations in rice plants associated with TALEN-based gene editing by investigating a number of morphological traits (plant height, tiller number, dry biomass, etc.). We have conducted a small scale field test in the ISU research farm with the five rice lines that had been ruled by the USDA APHIS BRS as non-regulated articles. The field test was performed in the summer of 2015 and is now ongoing this summer. From the last summer test, we did not find obvious negative effect of the SWEET gene promoter mutations on the overall plant growth and production of those mutant lines relative to the parent cultivar. Once this year's test is done, we will analyze the two years' data. We have completed 70% of the proposed aim. Impactful aspects of the results: Our results demonstrate that TALENs as one type of engineered nucleases represent a highly specific and efficient tool for crop improvement with the potential to rapidly and precisely generate useful novel genetic variations/traits. TALENs in particular, or engineered nucleases in general, as the mutagen for targeted genetic changes in plant cells and after completion of their task in one generation, can be sorted out through genetic cross and segregation in subsequent generations, resulting in transgene-free crop plants. The process seems different from the natural means or conventional mutagenesis, but the transgene-free products are not different. The TALEN-based product, if no transgenic DNA retained in the targeted genome, does not readily fit current definitions of genetically engineered or genetically modified organism (GMO) used within most regulatory regimes. Regulatory focus needs to be on the nature of the novel phenotype or product developed rather than on the process.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Si Nian Char, Anjanasree K. Neelakandan, Hartinio Nahampun, Bronwyn Frame, Marcy Main, Martin H. Spalding, Philip W. Becraft, Blake C. Meyers, Virginia Walbot, Kan Wang, Bing Yang. 2016. An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize. Plant Biotechnology Journal. First published online: 11 August 2016, DOI: 10.1111/pbi.12611.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Donald P. Weeks, Martin H. Spalding and Bing Yang. 2016. Use of designer nucleases for targeted gene and genome editing in plants. Plant Biotechnology Journal 14, 483495.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Jeffrey D. Wolt1, Kan Wang and Bing Yang. 2016. The Regulatory Status of Genome-edited Crops. Plant Biotechnology Journal 14, 510-518.
|
Progress 09/01/14 to 08/31/15
Outputs
Target Audience: The target audience includes the scientific community as our work has been presented as the invited talks in several scientific meetings and scientific publication, and also includes graduate/undergraduate students as our efforts reached classrooms in the forms of guest lectures and seminars in and out of ISU. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This grant has provide trainning opportunities for two graduate students and one undergraduate student as well as one scientist to work on the most advance biotechologies that provids promise in basic and applied science in agriculture. How have the results been disseminated to communities of interest? We have published one article and submitted another article for publicaton related to this project. The publication is one of the most efficient ways to disseminate our results among the scientific community. The PIs have also presented our effort and results in several scientific conferences and university lectures. For example, PI has presented the work and acknowledged the NIFA funding in the following conferences as the invited speaker: In Vitro Biology meeting, Tucson, Arizona, May-June, 2015 CROPS, Hutsville, Alabama, May, 2015 The 57th Annual Maize Genetics Conference, St. Charles, Illinois, 2015 The 12th International Symposium of Rice Functional Genomics, Tucson, Arizona, Nov., 2014 North American Agricultural Biotechnology Council 26, Ithaca, New York, Oct., 2014 The 4th International Rice Congress, Bangkok, Thailand, Oct., 2014 The 13th International Conference on Plant Pathogenic Bacteria, Shanghai, June, 2014 †Snips & Pics: From Genome Targeting to Imaging, a short course hosted by the Genes & Development Graduate Program at the University of Texas MD Anderson Cancer Center, Houston, January 29-30, 2014 What do you plan to do during the next reporting period to accomplish the goals? We will continue to work on the objectives we proposed based on the proposed timetable.
Impacts
What was accomplished under these goals?
1-1. We have established a pipeline of TALEN-based gene editing in rice. The pipeline includes (1) design and engineer any novel TALEN genes using a "Gold gate" modular assembly method with a TAL repeat library we have developed, (2) introduce TALEN constructs into rice embryogenic callus cells and generate transgenic rice lines through tissue culture and transformation, (3) identify TALEN-mediated gene editing in primary transgenic plants and their progeny, and (4) characterize the TALEN-mediated mutant plants molecularly and physiologically. To assess its robustness of this pipeline for TALEN-mediated gene editing, we chose 15 rice SWEET (sugar transporter) genes for TALEN-mediated gene editing. We have made 15 TALEN constructs each targeting one SWEET gene, and have obtained edited plants for 7 SWEET genes, but have not obtained edited plants from 8 TALEN constructs. For those constructs that generated gene editing, the frequency ranged from about 15% to 70%. 1-2. We have also established a workable protocol for switchgrass tissue culture and Agrobacterium-mediated transformation. Transformation of TALEN constructs targeting three switchgrass genes is in progress. 1-3. We have expanded our effort to suscessfully establish a CRISPR/Cas9 system for gene editing in rice (Zhou et al. Nucleic Acids Research 42:10903-10914) and also switchgrass. This endeavour will allow us to compare the two most advanced gene editing or mutagenesis technologies in rice and switchgrass. We have whole genome sequenced 8 rice lines including one parental line Kitaake and seven lines that contained the TALEN-mediated edit in the promoters of SWEET11 and SWEET14. The sequencing work with Illumina and PacBio technologies has been completed. The whole genome assembly of the eight genomes are still in progress. Analysis of the Illumina sequencing data indicates that five mutant lines do not contain any transgene of TALENs and transformation selection marker gene (hygromycin resistant gene), but two lines contain part of T-DNA. We have inquired the ruling from the USDA APHIS BRS on the five TALEN-mutagenized rice lines, and have been informed of non-regulated status of those lines. With this results, we will going to do a small scale of field trial in the Iowa State University research farm for morphological trait analysis in this upcoming summer.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Si Nian Char, Erica Unger-Wallace, Bronwyn Frame, Sarah A. Briggs, Marcy Main, Martin H. Spalding, Erik Vollbrecht,Kan Wang, Bing Yang. 2015. Heritable site-specific gene mutagenesis using TALENs in maize. Plant Biotechnology Journal. First published online: 3 Feb. 2015, DOI: 10.1111/pbi.12344.
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Progress 09/01/13 to 08/31/14
Outputs
Target Audience: The target audience includes the scientific community as our work has been presented as the invited talks in several scientific meetings and also includes graduate/undergraduate students as our efforts reached classrooms in the forms of guest lectures and seminars in and out of ISU. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This grant has provide trainning opportunities for two graduate students and one undergraduate student as well as one scientist to work on the most advance biotechologies that provids promise in basic and appliedscience inagriculture. How have the results been disseminated to communities of interest? We have published one article and submitted another article for publicaton related to this project. The publication is one of the most efficient ways to disseminate our results among thescientific community. The PIshave alsopresented our effortand results in several scientific conferences and university lectures. What do you plan to do during the next reporting period to accomplish the goals? We will continue to work on the objectives we proposed based on the proposed timetable.
Impacts
What was accomplished under these goals?
We have made progress in the following areas of the Major goals: 1. Establish a pipeline of TALEN-based gene editing and assess its robustness and general applicability in crop plants by focusing on rice and switchgrass. 1-1. We have established a pipeline of TALEN-based gene editing in rice. The pipeline includes (1) design and engineer any novel TALEN genes using a "Gold gate" modular assembly method with a TAL repeat library we have developed, (2) introduce TALEN constructs into rice embryogenic callus cells and generate transgenic rice lines through tissue culture and transformation, (3) identify TALEN-mediated gene editing in primary transgenic plants and their progeny, and (4) characterize the TALEN-mediated mutant plants molecularly and physiologically. To assess its robustness of this pipeline for TALEN-mediated gene editing, we chose15 rice SWEET (sugar transporter) genes for TALEN-mediated gene editing. We have made 15TALEN constructs each targeting one SWEET gene,and have obtained edited plants for 7 SWEET genes, but have not obtained edited plants from 8 TALEN constructs. For those constructs that generated gene editing, the frequency ranged from about15% to 70%. 1-2. We have also established a workable protocol for switchgrass tissue culture and Agrobacterium-mediated transformation. Construction of TALEN plasmids targeting three switchgrass genes is in progress. 1-3. We have expanded our effort to suscessfully establish a CRISPR/Cas9 system for gene editing in rice (Zhou et al. Nucleic Acids Research42:10903-10914). This endeavour will allow us to compare the two most advanced gene editing or mutagenesis technologies in rice and probably in switchgrass. 2. Assess potential off-target mutations caused by TALENs in modified rice genomes by comparing seven modified genomes against the parental reference genome. We have whole genome sequenced 8 rice lines including one parental line Kitaake and seven lines that contained the TALEN-mediated edit in the promoters of SWEET11 and SWEET14. The sequencing work with Illumina and PacBio technologies has been done and data analysis for off-target mutations caused by TALENs is in progress. 3. Assess the removal of the TALEN transgenes from the modified rice genome through genetic segregation at the genomic level. The assessment of the removal of TALEN-transgenes from the above mentioned 7 lines will be complishedwith analysis of whole genome sequencing data in Objective 2. 4. Analyze potential phenotypic variations in rice plants associated with TALEN-based gene editing by investigating a number of morphological traits (plant height, tiller number, dry biomass, etc.). The propagation of seed from those rice mutant lines for phenotypic variation analysis is in progress.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Si Nian Char, Erica Unger-Wallace, Bronwyn Frame, Sarah A. Briggs, Marcy Main, Martin H. Spalding, Erik Vollbrecht, Kan Wang, Bing Yang. Heritable site-specific gene mutagenesis using TALENs in maize. Plant Biotechnology Journal
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Zhou H, Liu B, Weeks DP, Spalding MH, Yang B (2014) Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Research 42(17):10903-10914.
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