Progress 09/01/19 to 08/31/23
Outputs
Target Audience: Target audiences include federal regulators, plant genetics and genomics researchers, geneticists and breeders, evolutionary biologists, and population geneticists. They also include users and producers of soybeans or other crops subject to transgenesis or other forms of genetic modification. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training and professional development opportunities for undergraduate students, a graduate student, and a postdoctoral scholar. All trainees involved in the project have been able to improve their biocomputing and programming skills and are learning data management and analysis skills. The lab also has a weekly lab meeting and journal club that provides the opportunity to discuss science and genetics informally with lab members. The grant supported graduate student Chaochih Liu, who has helped develop the computational tools to process large amounts of DNA sequencing data and for accurate variant calling and filtering. The grant contributed to her stipend support during a PhD program she completed in June 2023. She is now employed by Pepsi Co. Postdoctoral scholar Roland Akakpo, who also contributed to developing DNA sequencing handling pipelines. The grant has also supported three undergraduate students, Elaine Lee, Jacob Pacheco, and Nathan Liang. Ms. Lee and Mr. Pacheco are completing undergraduate degrees and continue to work with the lab. Mr. Liang graduated and works as a computer programmer for Well Fargo Bank. He continues to contribute to the data analysis related to this project. A new postdoctoral scholar, Willian Luis Arge Pacheco, was hired just as the grant ended. He is helping with the publication of results from the project. How have the results been disseminated to communities of interest?Dissemination of results from the project has mostly occurred through publications in the primary scientific literature. Two publications from the project have been reported previously, and three additional publications are in preparation. The project has also contributed to the development of computer code that is publicly available and is being used by researchers at several other institutions. This code is part of a workflow called "sequence_handling" that automates the processing of large amounts of resequencing data processing on a supercomputing cluster. We have also developed workflows and resources that characterize "callable" regions of the soybean genome, primarily by identifying genomic regions with high degrees of repetitive sequences or deletions relative to the reference. We have also developed code for manipulating long read (Oxford Nanopore) sequencing. These resources are noted in this report and available in public GitHub repositories. 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- Create CRISPR/Cas9 edited soybean families where lineages differ in the number of generations they carried the CRISPR/Cas9 transgene (Years 1 - 2). We have completed the generation of families of soybean plants that included transgenes with a CRISPR/Cas9 construct for eight different genes. The families are descended from the soybean varieties Bert and Williams82 (reference genome). Each family begins with a T0M0 plant that is targeted for transformation. We are currently growing out plants and carrying out DNA extractions for plants that differ in terms of at least two generations of exposure to a transgene. The potential for greater exposure to a CRISPR/Cas9 construct to create more off-target and unintended mutations is a central theme of this research and will improve understanding of the safe use of target genetic modifications. Under separate funding, co-PD Dr. Robert Stupar and colleagues have completed a "platinum quality" Willams82 reference genome, which will aid in accurately detecting off-target variants. The genome assembly and annotation incorporating the gene names used in previous builds of the Williams82 reference genome are now available through the Department of Energy's Joint Genome Institute website, Phytozome. We are moving all future analysis and resource-building to this improved genome build. Objective 2 - Use phenotypic screening and DNA resequencing to identify on-target, off-target, and genome-wide changes induced by CRISPR/Cas9 activity (Years 1 - 3). Phenotype screening has been ongoing for each family created. This has involved screening for "off-types" and potentially desirable phenotypes induced by CRISPR/Cas9 modification. We also need to use PCR-based screening for the presence of transgenes within transformed lines. CRISPR/Cas9 modifications and the creation of "families" of individuals descended from the initially transformed T0M0 plant have been completed for eight different soybean genes. PCR-based screening to identify plants that do or do not carry transgenes has been completed. We are sequencing the T0M0 plants using Illumina paired-end sequencing and the newer and less expensive Element AVITI sequencing technology. If the AVITI sequencing data can be used as a drop-in replacement, roughly halving the cost of sequencing will allow us to include more progeny from the individual families of soybean lines that differ in transgene exposure. Currently plans are to sequence between four to six individuals per family. We also proposed using Oxford Nanopore long-read sequencing to identify intended and unintended changes in duplicated genes. This portion of the project has taken on increased importance as it has become clear that CRISPR/Cas9 edits at multiple (duplicated) positions in the genome will often be necessary for crop species, especially polyploid species like soybean. We have completed Nanopore sequencing for the soybean varieties Bert and Williams82. This data is being used to create localized assemblies that will improve the characterization of gene targets, particularly at multiple copy genes in the soybean genome. The culmination of these efforts should result in at least three additional publications derived from this research effort.
Publications
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience: Target audiences include federal regulators, plant genetics and genomics researchers, geneticists and breeders, evolutionary biologists, and population geneticists. They also include users and producers of soybeans or other crops subject to transgenesis or other forms of genetic modification. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training and professional development opportunities for undergraduate students, a graduate student, and a postdoctoral scholar. All trainees involved in the project have been able to improve their biocomputing and programming skills and are learning data management and analysis skills. The lab also has a weekly lab meeting and journal club that provides the opportunity to discuss science and genetics informally with lab members. The grant supported graduate student Chaochih Liu, who has helped develop the computational tools to process large amounts of DNA sequencing data and for accurate variant calling and filtering. The grant contributed to her stipend support during a PhD program she completed in June 2023. She is now employed by Pepsi Co. Postdoctoral scholar Roland Akakpo, who also contributed to developing DNA sequencing handling pipelines. The grant has also supported three undergraduate students, Elaine Lee, Jacob Pacheco, and Nathan Liang. Ms. Lee and Mr. Pacheco are completing undergraduate degrees and continue to work with the lab. Mr. Liang graduated and works as a computer programmer for Well Fargo Bank. He continues to contribute to the data analysis related to this project. A new postdoctoral scholar, Willian Luis Arge Pacheco, was hired just as the grant ended. He is helping with the publication of results from the project. How have the results been disseminated to communities of interest?Dissemination of results from the project has mostly occurred through publications in the primary scientific literature. Two publications from the project have been reported previously, and three additional publications are in preparation. The project has also contributed to the development of computer code that is publicly available and is being used by researchers at several other institutions. This code is part of a workflow called "sequence_handling" that automates the processing of large amounts of resequencing data processing on a supercomputing cluster. We have also developed workflows and resources that characterize "callable" regions of the soybean genome, primarily by identifying genomic regions with high degrees of repetitive sequences or deletions relative to the reference. We have also developed code for manipulating long-read (Oxford Nanopore) sequencing. These resources are noted in this report and available in public GitHub repositories. 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- Create CRISPR/Cas9 edited soybean families where lineages differ in the number of generations they carried the CRISPR/Cas9 transgene (Years 1 - 2). We have completed the generation of families of soybean plants that included transgenes with a CRISPR/Cas9 construct for eight different genes. The families are descended from the soybean varieties Bert and Williams82 (reference genome). Each family begins with a T0M0 plant that is targeted for transformation. We are currently growing out plants and carrying out DNA extractions for plants that differ in terms of at least two generations of exposure to a transgene. The potential for greater exposure to a CRISPR/Cas9 construct to create more off-target and unintended mutations is a central theme of this research and will improve understanding of the safe use of target genetic modifications. Under separate funding, co-PD Dr. Robert Stupar and colleagues have completed a "platinum quality" Willams82 reference genome, which will aid in accurately detecting off-target variants. The genome assembly and annotation incorporating the gene names used in previous builds of the Williams82 reference genome are now available through the Department of Energy's Joint Genome Institute website, Phytozome. We are moving all future analysis and resource-building to this improved genome build. Objective 2 - Use phenotypic screening and DNA resequencing to identify on-target, off-target, and genome-wide changes induced by CRISPR/Cas9 activity (Years 1 - 3). Phenotype screening has been ongoing for each family created. This has involved screening for "off-types" and potentially desirable phenotypes induced by CRISPR/Cas9 modification. We also need to use PCR-based screening for the presence of transgenes within transformed lines. CRISPR/Cas9 modifications and the creation of "families" of individuals descended from the initially transformed T0M0 plant have been completed for eight different soybean genes. PCR-based screening to identify plants that do or do not carry transgenes has been completed. We are sequencing the T0M0 plants using Illumina paired-end sequencing and the newer and less expensive Element AVITI sequencing technology. If the AVITI sequencing data can be used as a drop-in replacement, roughly halving the cost of sequencing will allow us to include more progeny from the individual families of soybean lines that differ in transgene exposure. Currently plans are to sequence between four to six individuals per family. We also proposed using Oxford Nanopore long-read sequencing to identify intended and unintended changes in duplicated genes. This portion of the project has taken on increased importance as it has become clear that CRISPR/Cas9 edits at multiple (duplicated) positions in the genome will often be necessary for crop species, especially polyploid species like soybean. We have completed Nanopore sequencing for the soybean varieties Bert and Williams82. This data is being used to create localized assemblies that will improve the characterization of gene targets, particularly at multiple copy genes in the soybean genome. The culmination of these efforts should result in at least three additional publications derived from this research effort.
Publications
- Type:
Theses/Dissertations
Status:
Accepted
Year Published:
2023
Citation:
Liu C (2023) Biological problem solving through computation. Conservancy University of Minnesota https://hdl.handle.net/11299/259763
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Progress 09/01/20 to 08/31/21
Outputs
Target Audience:Target audiences include federal regulators, plant genetics and genomics researchers, geneticists and breeders, evolutionary biologists, and population geneticists. They also include users and producers of soybeans or other crops subject to transgenesis or other forms of genetic modification. Changes/Problems:Starting in March 2020 all University of Minnesota facilities were closed to all but essential functions. While that predates the current reporting period, we have not seen a return to full participation in on campus activities. Particularly during 2020, new laboratory work was limited. Undergraduate students working on the project have worked on campus much less frequently. This slowed but did not eliminate progress on this project. What opportunities for training and professional development has the project provided?The project has provided training and professional development opportunities for undergraduate students and graduate student. All students involved in the project have had an opportunity to improve their biocomputing skills and the opportunity to discuss science and genetics both informally with lab members and in lab meetings / journal club. The grant supported graduate student Chaochih Liu who has helped develop the computational tools we need to process large amounts of DNA sequencing data and for accurate variant calling and filtering of that data. The grant contributed to her stipend support during a PhD program. She is expected to complete her dissertation and degree in October 2022. The grant has also supported three undergraduate students, Elaine Lee, Nadia Janis, and Nathan Liang. Ms. Janis has graduated from the University of Minnesota and is working in environmental science related to mitigating climate change. Ms. Lee and Mr. Liang are continuing as undergraduate students in chemical engineering and computer science. Both students are gaining skills in programming along with data management and analysis. How have the results been disseminated to communities of interest?article on the genetic effects of targeted genetic changes while the second publication is focused on measuring the nucleotide sequence-level effects of traditional biotechnology approaches. The perspectives article involved co-PDs Stupar and Morrell and industry collaborators from four biotechnology companies. The paper on genetic variants helped develop components of the variant calling, filtering, and analysis tools used in the present study. These tools are made public through a GitHub repository. The workflow is being used by researchers at a number of other institutions. Code written for the analysis in this paper paper is available through a second GitHub repository also linked through this report. The publication is also associated with a repository with a unique digital object identifier (DOI) for files need for our analyses of genetic changing in soybean and our individual experimental lines. All of this reporting period occurred during the COVID-19 pandemic, so the co-PDs did not give public talks related to the project. What do you plan to do during the next reporting period to accomplish the goals?Our primary activity will be to continue to track and screen families of plants that do or do not carry transgenes with a CRISP/Cas9 construct. We will also continue phenotypic screening of these families. Because our primary focus is on the creation of families with different levels of exposure to the CRISPR/Cas9 construct, we also need to use PCR to screen lines for the transgenes. Ultimately these lines need to be maintained over generations to create populations for comparative resequencing. We have actively developed DNA extraction and library preparation approaches for Oxford Nanopore sequencing. We are now using these approaches to identify targeted and non-targeted mutations particularly in relation to duplicated genes. Based on our experience, ~10x coverage of the soybean genome is possible in a single flow cell run. This coverage should be sufficient to detect targeted changes.
Impacts
What was accomplished under these goals?
Objective 1- Create CRISPR/Cas9 edited soybean families where lineages differ in the number of generations they carried the CRISPR/Cas9 transgene (Years 1 - 2). 60% complete - We continued the generation of families of soybean plants that included a transgene with a CRISPR/Cas9 construct. As noted in the previous year, most lines are generated in the Bert and Jack genetic backgrounds, though lines in the Williams82 (reference genome) background are also being produced. These lines should make for easier detection of off-target mutations. This detection process should be improved by a "platinum quality" Williams82 reference genome being completed by Dr. Robert Stupar and colleagues under separate funding. Objective 2 - Use phenotypic screening and DNA resequencing to identify on-target, off-target, and genome-wide changes induced by CRISPR/Cas9 activity (Years 1 - 3). 60% complete - Phenotype screening has been ongoing for each family created. This has involved screening for "off-types" and potentially desirable phenotypes induced by CRISPR/Cas9 modification. We also need to use PCR-based screening for the presence of transgenes within transformed lines. Ultimately our approach involves screening for genome-wide changes to identify genetic (heritable) changes due to increased exposure to the CRISPR/Cas9. Our experiment is designed to permit the isolation of the role of CRISPR/Cas9 in creating these changes because we are creating families with increasing numbers of generations of exposure to the transgene. Thus the DNA sequencing and screening for variants are planned for later stages of the experiment. We also proposed the use of Oxford Nanopore long-read sequencing for the identification of both intended and unintended changes in duplicated genes. This portion of the project has taken on increased importance as it has become clear that CRISPR/Cas9 edits at multiple (duplicated) positions in the genome will often be necessary for crop species, especially polyploid species like soybean.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Graham N, Patil G, Bubeck DM, Dobert RC, Glenn KC, Gutsche AT, Kumar S, Lindbo JA, Maas L, May GD, Vega-Sanchez ME, Stupar RM, Morrell PL (2020) Plant genome editing and the relevance of off-target changes. Plant Physiology 183: 1453-1471. doi: 10.1104/pp.19.01194
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Wyant SR, Rodriguez MF, Carter CK, Parrott WA, Jackson SA, Stupar RM, Morrell PL (2021) Fast neutron mutagenesis in soybean enriches for small indels and creates frameshift mutations. G3: Genes Genomes Genetics doi: 10.1093/g3journal/jkab431
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Progress 09/01/19 to 08/31/20
Outputs
Target Audience:Target audiences include federal regulators, plant genetics and genomics researchers, geneticists and breeders, evolutionary biologists, and population geneticists. They also include users and producers of soybeans or other crops subject to transgenesis or other forms of genetic modification. Changes/Problems:Starting in March 2020 and continuing through the end of this reporting period all University of Minnesota facilities were closed to all but essential functions. The care and maintenance of plant tissue culture and plant growth facilities were covered as essential activities but most new laboratory work was limited. Undergraduate students working on the project also had limited access to campus facilities. This slowed but did not eliminate progress on this project. What opportunities for training and professional development has the project provided?The project has provided training and professional development opportunities for undergraduate students and a graduate student. All students involved in the project have had an opportunity to improve their biocomputing skills and the opportunity to discuss science and genetics both informally with lab members and in lab meetings / journal club. The grant supported undergraduate Mackenzie Linane, who worked primarily on sample organization and data submission. Ms. Linane graduated from the University of Minnesota and is currently studying for a Doctor of Osteopathic Medicine degree. How have the results been disseminated to communities of interest?Computational resources being developed as a portion of this project are being made available on a public GitHub repositories, including https://github.com/MorrellLAB/sequence_handling. The workflow is being used by researchers at a number of other institutions. Much of this reporting period occurred during the COVID-19 pandemic, so the co-PDs did not give public talks related to the project. ? What do you plan to do during the next reporting period to accomplish the goals?Our primary activity will be to continue to track and screen families of plants that do or do not carry transgenes with a CRISP/Cas9 construct. We will also continue phenotypic screening of these families. Because our primary focus is on the creation of families with different levels of exposure to the CRISPR/Cas9 product, we also need to use PCR to screen lines for the transgenes. Ultimately these lines need to be maintained over generations to create populations for comparative resequencing.
Impacts
What was accomplished under these goals?
Objective 1- Create CRISPR/Cas9 edited soybean families where lineages differ in the number of generations they carried the CRISPR/Cas9 transgene (Years 1 - 2). 30% complete - We began the generation of families of individuals with CRIPSR/Cas9 transgenes inserted. The initial transgenic lines were created in the Bert and Jack backgrounds, as planned. In addition, the laboratory of Dr. Robert Stupar has begun to create transgenic lines using Williams82, the same background which was used to create the soybean reference genome. Transformation of this line provides the advantage of minimizing the number of differences between the transformed line and the reference genome. For our purposes, this makes the detection of off-target CRISPR/Cas9 changes more tractable. We proposed the creation of 15 families. We plan to stick to this plan, but with some families created as sublineages of a transformation, owing to the challenges of creating segregating families. This plan should allow us to meet our proposed aims. Objective 2 - Objective 2. Use phenotypic screening and DNA resequencing to identify on-target, off-target, and genome-wide changes induced by CRISPR/Cas9 activity (Years 1 - 3). 30% complete - Phenotype screening has been ongoing for each of the families created. This has involved screening for both "off-types" and for potentially desirable phenotypes induced by CRISPR/Cas9 modification. This also requires PCR-based screening for the presence of transgenes within transformed lines. Screening for genome-wide changes was proposed as a means of identifying genetic (heritable) changes due to increased exposure to the CRISPR/Cas9. Our experimental design permits the isolation of the role of CRISPR/Cas9 in creating these changes because we are creating families with different numbers of generations of exposure to the transgene. Thus the DNA sequencing and screening for variants are planned for later stages of the experiment.
Publications
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