Progress 07/01/20 to 06/30/23
Outputs
Target Audience:The project outputs were aimed towards the scientific community working towards developing more efficient biotechnology tools for crop improvement, including faster plant transformation and gene editing protocols. Across the entire project, wehave published our results in sevenpeer-reviewed journal articles to reach the target audience with our optimized protocols (see Products). Changes/Problems:The lab faced significant challenges with local COVID-19 spikes in the summer of 2020, the winter of 2020/2021, and to a lesser extent the summer of 2021 and the winter of 2021/2022. Although the lab was never shut down due to its "essential services" status, we had to stagger work schedules to limit the number of students and staff in the lab at any one time. Moreover, sporadic delays in obtaining certain lab supplies also slowed down the research. In spite of these difficulties, we largely completed our objectives by the end of the 3rd year no-cost extension. What opportunities for training and professional development has the project provided?The following students and staff have been trained by this project in molecular biology techniques to optimize plant transformation and gene editing approaches: - Tia Dunbar: Master's student under Dr. Thomson's supervision; she took the lead on the rice CNT delivery experiments and graduated in December 2021. - Dr. Sudip Biswas: PhD student under Dr. Septiningsih's supervision, he graduated in May 2022 and stayed on as a post-doctoral researcher, focusing on peanut and rice. - Aya Bridgeland: MS student under Dr. Septiningsih's supervision; she took the lead on the cowpea CNT delivery experiments and graduated in May 2022. - Merve Saglam: PhD student under Dr. Septiningsih's supervision; she continued the work with the cowpea CNT delivery experiments. - Dr. Nikolaos Tsakirpaloglou: post-doctoral research associate with Dr. Thomson; he helped supervise the graduate students. How have the results been disseminated to communities of interest?The results have been disseminated through six conference papers and presentations and seven peer-reviewed journal articles published during this reporting period (see Products). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1 was to develop visible gene editing assays across each target crop. For rice, tissue culture and transformation of Presidio, a recalcitrant tropical japonica rice cultivar, was optimized using both immature embryos and mature seeds, and a visible gene editing assay based on a knockout of the rice PDS gene was validated and published (Molina-Risco et al. 2021). In peanut, the guide RNA validation for two PDS copies in the tetraploid peanut produced ambiguous results from the Agrobacterium-mediated transformation. Subsequently, a protoplast isolation protocol was optimized for peanut to allow for faster validation of guide RNAs and was successfully used for CRISPR/Cas9 editing of the allergen gene Ara h2 (Biswas et al. 2022). For cowpea, four gRNAs targeting the single copy PDS gene were designed and validated using the in vitro RNA assay. These gRNAs were inserted into two vectors: a binary vector for testing with Agrobacterium-meditated transformation, and a non-binary vector for delivery into protoplasts and for testing carbon nanotube-mediated delivery. A protocol to isolate protoplasts from cowpea leaves was optimized and protoplasts were transformed with the PDS gRNAs, successfully producing target mutations (Bridgeland et al. 2023). In sorghum, four gRNAs were designed to target the single copy PDS gene in the sorghum genome, and 3 out of 4 were validated with the in vitro RNP assay. Several promoters were tested with GFP to determine optimal gene expression, and a transformation construct with the 3 gRNAs was developed. The sorghum results have not yet been published. Objective 2 aimed to optimize in planta transformation protocols for rice and peanut. To test carbon nanotube (CNT) based delivery in rice, we started by using PEI functionalization to attach plasmid DNA to carbon nanotubes (pDNA-CNTs). We used GFP and GUS as reporter genes to test whether the pDNA-CNTs can enter rice tissues. Rice leaf infiltrations were tested by puncturing leaves with needles and soaking in a pDNA-CNT solution for several days. Excised embryos from germinated rice seeds were also tread in a pDNA-CNT solution. GFP and GUS results showed delivery of the plasmids into the rice leaves and seeds. Next, a plasmid construct containing Cas9 + PDS-gRNAs was attached to CNTs and used to treat both imbibed whole seeds and excised embryos, and sequence analysis showed some possible mutations at the target site. These results were recently published (Dunbar et al. 2022). To test pDNA-CNT delivery into peanut leaves, several leaf infiltration experiments were performed using the needle-less syringe approach, with testing different ratios of plasmid DNA to CNTs, testing two different promoters, and evaluating both GFP and GUS expression. GFP fluorescence had some challenges due to a low level of background fluorescence, but the GUS assays confirmed successful delivery of plasmid DNA into peanut leaf tissue using CNTs. Delivery into imbibed peanut seeds was also tested, but with ambiguous results. Although the CNT delivery using peanut was not fully optimized, the project worked to test the new method of prime editing in peanut and other crops, and successfully showed prime editing as working in peanut protoplasts (Biswas et al. 2023). Objective 3 worked to test the optimized in planta transformation protocol across all four target crops. Overall, the CNT delivery was successful in rice, as seen in the Dunbar et al 2022 publication. CNT delivery for gene editing was also demonstrated in cowpea (Saglam et al. 2023 abstract presented at the CSSA Annual Meeting; manuscript is in preparation). Initial experiments with cowpea focused on testing pDNA-CNT delivery using leaf infiltrations with GFP and GUS. GFP expression was visible immediately around the infiltration site, while the GUS expression was detected in the infiltrated leaf as well as the surrounding vascular tissue, suggesting that the pDNA-CNTs may be traveling through the plant. Subsequent experiments with using CNT delivery for CRISPR/Cas9-mediated genome editing detected deletions at the PDS target gene, indicating successful delivery (Saglam et al. 2023 conference abstract). The results in peanut and sorghum had issues that suggested there were species-specific differences that would have to be overcome before CNT delivery can be used more widely for plant transformation and gene editing. The project was also highlighted in two review articles produced by the PI, Co-PI, and our post-doctoral research associates: "Functional allele validation by gene editing to leverage the wealth of genetic resources for crop improvement" which discussed the latest advances tested in this project, including validation using protoplasts, leaf infiltration, and in planta transformation with carbon nanotubes (Thomson et al. 2022). A second review presented "Guidelines for performing CRISPR/Cas9 genome editing for gene validation and trait improvement in crops" and discussed best practices and lessons learned from this project, including guide RNA design, construct prepration, plant transformation, and detection of mutations (Tsakirpaloglou et al. 2023).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Biswas S, Bridgeland A, Irum S, Thomson MJ, Septiningsih EM (2022) Optimization of prime editing in rice, peanut, chickpea, and cowpea protoplasts by restoration of GFP activity. Int. J. Mol. Sci. 23:9809.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Bridgeland A, Biswas S, Tsakirpaloglou N, Thomson MJ, Septiningsih EM (2023) Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene. PLoS ONE 18: e0283837.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Tsakirpaloglou N, Septiningsih EM, Thomson MJ (2023) Guidelines for performing CRISPR/Cas9 genome editing for gene validation and trait improvement in crops. Plants 12:3564.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Saglam M, Bridgeland A, Tsakirpaloglou N, Thomson, MJ, Septiningsih EM (2023) Carbon nanotube-mediated plasmid DNA delivery in cowpea leaves [Abstract]. ASA, CSSA, SSSA International Annual Meeting, St. Louis, MO. https://scisoc.confex.com/scisoc/2023am/meetingapp.cgi/Paper/153191
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Progress 07/01/21 to 06/30/22
Outputs
Target Audience:In this reporting period, the project outputs were aimed towards the scientific community working towards developing more efficient biotechnology tools for crop improvement, including faster plant transformation and gene editing protocols. We have published our results in four peer-reviewed journal articles to reach the target audience with our optimized protocols (listed as Products below). Changes/Problems:The lab faced challenges with local COVID-19 spikes in the summer of 2020, the winter of 2020/2021, and to a lesser extent the summer of 2021 and the winter of 2021/2022. Although the lab was never shut down due to its "essential services" status, we had to stagger work schedules to limit the number of students and staff in the lab at any one time. Moreover, sporadic delays in obtaining certain lab supplies also slowed down the research. In spite of these difficulties, we still hope to complete our objectives by the end of the 3rd year no-cost extension. What opportunities for training and professional development has the project provided?The following students and staff have been trained by this project in molecular biology techniques to optimize plant transformation and gene editing approaches: - Tia Dunbar: Master's student under Dr. Thomson's supervision; she took the lead on the rice CNT delivery experiments and graduated in December 2021. - Sudip Biswas: PhD student under Dr. Septiningsih's supervision, he graduated in May 2022 and stayed on as a post-doctoral researcher; he is leading the experiments on the peanut CNT delivery. - Aya Bridgeland: MS student under Dr. Septiningsih's supervision; she took the lead on the cowpea CNT delivery experiments and graduated in May 2022. - Merve Saglam: PhD student under Dr. Septiningsih's supervision; she is continuing the cowpea CNT delivery experiments. - Dr. Nikolaos Tsakirpaloglou: post-doctoral research associate with Dr. Thomson; helping to supervise the graduate students in plasmid construct development; he will continue the rice and sorghum CNT experiments. How have the results been disseminated to communities of interest?The results have been disseminated primarily through four peer-reviewed journal articles published during this reporting period (see Products). In addition to three research articles, one review article (Thomson et al. 2022) included a high-level overview of "Enabling Technologies" for crop gene editing, including validation with protoplasts, in planta transformation, carbon nanotube-based delivery, and the use of developmental regulators. What do you plan to do during the next reporting period to accomplish the goals?Due to the challenges listed below, we required a no-cost extension for an additional year after the original 2-year project. For this 3rd year, we plan to complete the experiments with imbibed seeds, and then pursue tests on pollen grains and immature flowers. We expect to have validated at least one in planta transformation approach for each of our four target crops by the conclusion of the 3-year project.
Impacts
What was accomplished under these goals?
Objective 1: Develop visible gene editing assays across each target crop Rice: For this objective, tissue culture and transformation of Presidio, a recalcitrant tropical japonica rice cultivar, was optimized using both immature embryos and mature seeds, and a visible gene editing assay based on a knockout of the rice PDS gene was validated and published (Molina-Risco et al. 2021). This system is now available to test the carbon nanotube-based delivery for gene editing in rice. Peanut: The guide RNA validation for two PDS copies in the tetraploid peanut produced ambiguous results from the Agrobacterium-mediated transformation. Subsequently, a protoplast isolation protocol was optimized for peanut to allow for faster validation of guide RNAs and was successfully used for CRISPR/Cas9 editing of the allergen gene Ara h2 (Biswas et al. 2022). Cowpea: After sequencing the single copy PDS gene in cowpea, four gRNAs were designed and validated using the in vitro RNA assay. These gRNAs were inserted into two vectors: a binary vector for testing with Agrobacterium-meditated transformation, and a non-binary vector for delivery into protoplasts and for testing carbon nanotube-mediated delivery. A protocol to isolate protoplasts from cowpea leaves was optimized and protoplasts were transformed with the PDS gRNAs, successfully producing target mutations (manuscript in preparation). Sorghum: Four gRNAs were designed to target the single copy PDS gene in the sorghum genome, and 3 out of 4 were validated with the in vitro RNP assay. Several promoters were tested with GFP to determine optimal gene expression, and a transformation construct with the 3 gRNAs was developed. Transformation and analysis is ongoing. Objective 2: Optimize in planta transformation protocols for rice and peanut Rice: To test carbon nanotube (CNT) based delivery in rice, we started by reproducing the Demirer et al. 2019 protocol to use PEI functionalization to attach plasmid DNA to carbon nanotubes (pDNA-CNTs). We used GFP and GUS as reporter genes to test whether the pDNA-CNTs can enter rice tissues. Rice leaf infiltrations were tested by puncturing leaves with needles and soaking in a pDNA-CNT solution for several days. Excised embryos from germinated rice seeds were also tread in a pDNA-CNT solution. GFP and GUS results showed delivery of the plasmids into the rice leaves and seeds. Next, a plasmid construct containing Cas9 + PDS-gRNAs was attached to CNTs and used to treat both imbibed whole seeds and excised embryos, and sequence analysis showed some possible mutations at the target site. These results were recently published (Dunbar et al. 2022). Peanut: To test pDNA-CNT delivery into peanut leaves, several leaf infiltration experiments were performed using the needle-less syringe approach, with testing different ratios of plasmid DNA to CNTs, testing two different promoters, and evaluating both GFP and GUS expression. GFP fluorescence had some challenges due to a low level of background fluorescence, but the GUS assays confirmed successful delivery of plasmid DNA into peanut leaf tissue using CNTs. Delivery into imbibed peanut seeds was also tested, and the GFP results looked promising, but need further confirmation. Objective 3: Test the optimized in planta transformation protocol across all four target crops Cowpea: As with rice and peanut, initial experiments with cowpea focused on testing pDNA-CNT delivery using leaf infiltrations with GFP and GUS. GFP expression was visible immediately around the infiltration site, while the GUS expression was detected in the infiltrated leaf as well as the surrounding vascular tissue, suggesting that the pDNA-CNTs may be traveling through the plant. Sorghum: Initial leaf infiltrations into young sorghum leaves using the needle-less syringe method showed promising results with GFP expression, but further experiments are needed to replicate these results.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Molina-Risco M, Ibarra O, Faion-Molina M, Kim B, Septiningsih EM, Thomson MJ (2021) Optimizing Agrobacterium-mediated transformation and CRISPR-Cas9 gene editing in the tropical japonica rice variety Presidio. Int. J. Mol. Sci. 22: 10909.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Biswas S, Wahl NJ, Thomson MJ, Cason JM, McCutchen BF, Septiningsih EM (2022). Optimization of protoplast isolation and transformation for a pilot study of genome editing in peanut by targeting the allergen gene Ara h 2. Int. J. Mol. Sci. 23: 837.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Dunbar T, Tsakirpaloglou N, Septiningsih EM, Thomson MJ (2022) Carbon Nanotube-Mediated Plasmid DNA Delivery in Rice Leaves and Seeds. Int. J. Mol. Sci. 23: 4081.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Thomson MJ, Biswas S, Tsakirpaloglou N, Septiningsih EM (2022) Functional allele validation by gene editing to leverage the wealth of genetic resources for crop improvement. Int. J. Mol. Sci. 23: 6565.
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Progress 07/01/20 to 06/30/21
Outputs
Target Audience:In the first year, the project outputs were primarily aimed towards the scientific community working towards developing more efficient biotechnology tools for crop improvement, including faster plant transformation and gene editing protocols. We have presented our initial results, which focused on optimizing PDS as a visible control for gene editing and testing carbon nanotubes as a novel delivery system, in several scientific conferences as poster and oral presentations (listed as Products below). Changes/Problems:The lab faced challenges with local COVID-19 spikes in the summer of 2020 and winter of 2020/2021. Although the lab was never shut down due to its "essential services" status, we had to stagger work schedules to limit the number of students and staff in the lab at any one time. Moreover, sporadic delays in obtaining certain lab supplies also slowed down the research. Despite these challenges, we were able to make significant progress in the first year and expect to continue to scale up our activities during the second year of the project to achieve the objectives. What opportunities for training and professional development has the project provided?The following students and staff have been trained by this project in molecular biology techniques to optimize plant transformation and gene editing approaches: - Oneida Ibarra: research associate with Dr. Thomson; initially worked on rice PDS and sorghum leaf infiltrations, then moved to another job in January 2021. - Tia Dunbar: Master's student under Dr. Thomson's supervision; she has been taking the lead on the rice CNT delivery experiments. - Sudip Biswas: PhD student under Dr. Septiningsih's supervision; he is leading the experiments on the peanut CNT delivery. - Aya Bridgeland: MS student under Dr. Septiningsih's supervision; she is leading the cowpea CNT delivery experiments. - Dr. Nikolaos Tsakirpaloglou: post-doctoral research associate with Dr. Thomson; helping to supervise the graduate students in plasmid construct development; will continue the sorghum CNT experiments. How have the results been disseminated to communities of interest?Thus far, the preliminary results have been primarily disseminated through conference presentations: Sudip presented two virtual posters on the peanut research (ASPB and CSSA meetings), Aya presented a virtual poster on the cowpea research (ASPB), the Co-PI Septiningsih gave an oral presentation covering both the peanut and cowpea results (CSSA), and Dr. Tsakirpaloglou presented a virtual poster giving an overview of the lab's gene editing activities (CSSA). In addition, the Co-PI Septiningsih gave a seminar to the Texas A&M Soil and Crop Sciences Department on genome editing activities in rice and peanut in October 2020, and Dr. Tsakirpaloglou gave a seminar to the Texas A&M Plant Breeding Circle on the gene editing pipeline in February 2021. What do you plan to do during the next reporting period to accomplish the goals?For the second year of the project, we plan to build on our initial success seen by the pDNA-CNT delivery through leaf infiltrations and proceed with testing in planta gene editing using the PDS knockouts as a visible phenotype. Experiments with imbibed seeds and SAMs have been initiated, and these will be followed by tests on pollen grains and immature flowers. We expect to have optimized at least one in planta transformation approach for each of our four target crops by the conclusion of the 2-year project.
Impacts
What was accomplished under these goals?
Objective 1: Develop visible gene editing assays across each target crop Rice: A portion of the rice PDS gene was sequenced in the Texas rice variety Presidio to compare with the Nipponbare reference genome, and two guide RNAs (gRNAs) were designed and validated by the Cas9 in vitro ribonucleoprotein (RNP) assay. These two gRNAs were inserted into binary and non-binary constructs and used for Agrobacterium- and particle bombardment-mediated rice transformation. Thirty albino plants were obtained and 18 were analyzed with Sanger sequencing at the two gRNA locations, revealing successful CRISPR/Cas9 editing of the PDS gene through insertions, deletions, and base substitutions (manuscript in preparation). Through this process, the tissue culture and transformation of Presidio, a recalcitrant tropical japonica rice cultivar, was optimized using both immature embryos and mature seeds, and this PDS knockout system is now available to test the carbon nanotube-based delivery for gene editing. Peanut: Portions of exons 2, 8 and 12 across the two PDS copies in the tetraploid peanut were sequenced, and three guide RNAs (gRNAs) were designed at conserved regions predicted to simultaneously knock out both PDS copies (A and B genomes). All three were validated by the in vitro RNP assay, and Agrobacterium-mediated transformation is currently ongoing to confirm that the gRNAs can be used to knock out both PDS copies in peanut. At the same time, we are also testing several different promoters to help increase the efficiency of gene editing in peanut. Cowpea: After sequencing the single copy PDS gene in cowpea, four gRNAs were designed (three in exon 2 and one in exon 3) and validated using the in vitro RNA assay. These gRNAs were inserted into two vectors: a binary vector for testing with Agrobacterium-meditated transformation, and a non-binary vector for delivery into protoplasts and for testing carbon nanotube-mediated delivery. To validate the gRNAs in vivo, a protocol to isolate protoplasts from cowpea leaves was optimized and protoplasts were transformed and sequence analysis is ongoing. Sorghum: Four gRNAs were designed to target the single copy PDS gene in the sorghum genome, and 3 out of 4 were validated with the in vitro RNP assay. Several promoters were tested with GFP to determine optimal gene expression, and a transformation construct with the 3 gRNAs was developed. Initial transformations using Agrobacterium- and particle bombardment-mediated delivery were made, and the analysis is ongoing. Objective 2: Optimize in planta transformation protocols for rice and peanut Rice: To test carbon nanotube (CNT) based delivery, we started by reproducing the Demirer et al. 2019 protocol to use PEI functionalization to attach plasmid DNA to carbon nanotubes (pDNA-CNTs). We initially used GFP and GUS as reporter genes to test whether the pDNA-CNTs can enter rice tissues. Rice leaf infiltrations were tested by puncturing leaves with needles and soaking in a pDNA-CNT solution for several days. Results from GFP had some challenges with auto-fluorescence from the leaf wounding but showed an increase in fluorescence 3 days post-infiltration with the pDNA-CNTs versus the negative control. The GUS results were more conclusive, with clear expression in the rice leaf midrib in the treated leaves versus the negative control, demonstrating successful passive delivery of pDNA-CNTs into rice leaf tissues. Next, excised embryos from germinated rice seeds were used to expose the shoot apical meristems (SAMs), which were treated in a pDNA-CNT solution. Again, GFP plasmids showed an increase in fluorescence, although these results need to be further confirmed. Subsequently, a plasmid construct containing Cas9 + PDS-gRNAs was attached to CNTs and used to treat both imbibed whole seeds and excised SAMs, and seedlings with partial albino phenotypes are currently undergoing sequence analysis. Peanut: To test pDNA-CNT delivery into peanut leaves, several leaf infiltration experiments were performed using the needle-less syringe approach, with testing different ratios of plasmid DNA to CNTs, testing two different promoters, and evaluating both GFP and GUS expression. Again, the GFP fluorescence had some challenges due to a low level of background fluorescence, but the GUS assays confirmed successful delivery of plasmid DNA into peanut leaf tissue using CNTs. Delivery into imbibed peanut seeds was also tested, and the GFP results looked promising, but need further confirmation. Objective 3: Test the optimized in planta transformation protocol across all four target crops Cowpea: As with rice and peanut, initial experiments with cowpea focused on testing pDNA-CNT delivery using leaf infiltrations with GFP and GUS. GFP expression was visible immediately around the infiltration site, while the GUS expression was detected in the infiltrated leaf as well as the surrounding vascular tissue, suggesting that the pDNA-CNTs may be traveling through the plant. Sorghum: Initial leaf infiltrations into young sorghum leaves using the needle-less syringe method showed promising results with GFP expression, but further experiments are needed to replicate these results.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Biswas S, Molina-Risco M, Faion-Molina M, Bellinatti-Della Gracia M, Thomson MJ, Septiningsih EM. 2020. Targeted mutagenesis of the phytoene desaturase using CRISPR/Cas9 multiplex genome editing system in allotetraploid peanut. PB20 Plant Biology Worldwide Summit, Virtual, 27-31 July 2020 (poster)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Bridgeland A, Tsakirpaloglou N, Thomson M, Septiningsih E. 2020. Genome editing optimization in cowpea using CRISPR/Cas9. PB20 Plant Biology Worldwide Summit, Virtual, 27-31 July 2020 (poster)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Tsakirpaloglou N, Ibarra O, Septiningsih EM, Thomson MJ. 2020. Optimizing a high-throughput gene editing pipeline at the Texas A&M Crop Gene Editing Lab. PB20 Plant Biology Worldwide Summit, Virtual, 27-31 July 2020 (poster)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Biswas S, Molina-Risco M, Faion-Molina M, Thomson MJ, Septiningsih EM. 2020. Efficiently knockout of the phytoene desaturase gene in peanut by the CRISPR/Cas9 genome editing system. Crop Science Society of America, Virtual, 9-13 November 2020 (poster).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Septiningsih EM, Biswas S, Bridgeland A, Molina-Risco M, Faion-Molina M, Tsakirpaloglou N, Thomson M. 2020. Optimization of CRISPR/Cas9 genome editing in peanut and cowpea. Crop Science Society of America, Virtual, 9-13 November 2020 (oral presentation).
NIFA Support Acknowledged. YES
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