An Undergraduate Research Project Utilizing CRISPR-Cas9 Gene Editing Technology to Study Gene Function in Arabidopsis thaliana †
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Authors:
Nicholas J. Ruppel1,
Lauren E. Estell1,
Robert I. Jackson2,
Michael J. Wolyniak2
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Received 31 July 2018 Accepted 26 February 2019 Published 28 June 2019
- ©2019 Author(s). Published by the American Society for Microbiology
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[open-access] This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/ and https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode), which grants the public the nonexclusive right to copy, distribute, or display the published work.
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†Supplemental materials available at http://asmscience.org/jmbe
- *Corresponding author. Mailing address: Randolph-Macon College, P.O. Box 5005, Ashland VA, 23005. Phone: 804-752-7267. E-mail: [email protected].
Abstract:
The CRISPR-Cas9 system functions in microbial viral pathogen recognition pathways by identifying and targeting foreign DNA for degradation. Recently, biotechnological advances have allowed scientists to use CRISPR-Cas9-based elements as a molecular tool to selectively modify DNA in a wide variety of other living systems. Given the emerging need to bring engaging CRISPR-Cas9 laboratory experiences to an undergraduate audience, we incorporated a CRISPR-based research project into our Genetics class laboratories, emphasizing its use in plants. Our genetic manipulations were designed for Arabidopsis thaliana, which despite serving as a plant research model, has traditionally been difficult to use in a classroom setting. For this project, students transformed plasmid DNA containing the essential CRISPR-Cas9 gene editing elements into A. thaliana. Expression of these elements in the plant genome was expected to create a deletion at one of six targeted genes. The genes we chose had a known seedling and/or juvenile loss-of-function phenotype, which made genetic analysis by students with a limited background possible. It also allowed the project to reach completion in a typical undergraduate semester timeframe. Assessment efforts demonstrated several learning gains, including students’ understanding of CRISPR-Cas9 content, their ability to apply CRISPR-Cas9 gene editing tools using bioinformatics and genetics, their ability to employ elements of experimental design, and improved science communication skills. They also felt a stronger connection to their scientific education and were more likely to continue on a STEM career path. Overall, this project can be used to introduce CRISPR-Cas9 technology to undergraduates using plants in a single-semester laboratory course.
References & Citations
Supplemental Material
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Appendix 1: Materials, Appendix 2: Plant transformation protocol, Appendix 3: Plant DNA isolation and PCR amplification protocol, Appendix 4: Bioinformatics exercise I, Appendix 5: Bioinformatics exercise II, Appendix 6: Bioinformatics exercise I – student example, Appendix 7: Agrobacterium tumefaciens transformation protocol, Appendix 8: Agrobacterium tumefaciens colony PCR protocol, Appendix 9: Final written paper – student example, Appendix 10: Final written paper rubric, Appendix 11: Oral presentation rubric, Appendix 12: Pre- and post-content assessment survey
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Abstract:
The CRISPR-Cas9 system functions in microbial viral pathogen recognition pathways by identifying and targeting foreign DNA for degradation. Recently, biotechnological advances have allowed scientists to use CRISPR-Cas9-based elements as a molecular tool to selectively modify DNA in a wide variety of other living systems. Given the emerging need to bring engaging CRISPR-Cas9 laboratory experiences to an undergraduate audience, we incorporated a CRISPR-based research project into our Genetics class laboratories, emphasizing its use in plants. Our genetic manipulations were designed for Arabidopsis thaliana, which despite serving as a plant research model, has traditionally been difficult to use in a classroom setting. For this project, students transformed plasmid DNA containing the essential CRISPR-Cas9 gene editing elements into A. thaliana. Expression of these elements in the plant genome was expected to create a deletion at one of six targeted genes. The genes we chose had a known seedling and/or juvenile loss-of-function phenotype, which made genetic analysis by students with a limited background possible. It also allowed the project to reach completion in a typical undergraduate semester timeframe. Assessment efforts demonstrated several learning gains, including students’ understanding of CRISPR-Cas9 content, their ability to apply CRISPR-Cas9 gene editing tools using bioinformatics and genetics, their ability to employ elements of experimental design, and improved science communication skills. They also felt a stronger connection to their scientific education and were more likely to continue on a STEM career path. Overall, this project can be used to introduce CRISPR-Cas9 technology to undergraduates using plants in a single-semester laboratory course.

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Author and Article Information
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Received 31 July 2018 Accepted 26 February 2019 Published 28 June 2019
- ©2019 Author(s). Published by the American Society for Microbiology
-
[open-access] This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial-NoDerivatives 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/ and https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode), which grants the public the nonexclusive right to copy, distribute, or display the published work.
-
†Supplemental materials available at http://asmscience.org/jmbe
- *Corresponding author. Mailing address: Randolph-Macon College, P.O. Box 5005, Ashland VA, 23005. Phone: 804-752-7267. E-mail: [email protected].
Figures

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FIGURE 1
The workflow for each weekly unit during a 14-week semester. The two bioinformatic exercises ( Appendices 4 and 5 ) are labeled as Web 1 and Web 2. Our spring break occurred during the 8th week when T1 transgenic plants were growing. CRISPR = clustered regularly interspaced short palindromic repeat.

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FIGURE 2
Image of TMM T1 transgenic plant. Note that most of the T1 generation plants were susceptible to the glufosinate-ammonium spray. Scale equals 1 cm.

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FIGURE 3
Pre- and posttest content assessment results. The quiz had 11 questions ( Appendix 12 ) and was administered in the first and final laboratory periods. Student scores improved from an average of 4.61±0.19 to 5.8±0.29 (by Student’s t-test, p<0.01, n=41). Bars represent standard error.

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FIGURE 4
Hypocotyl length of seven-day-old wild-type A. thaliana and the HY5 mutant (ABRC CS71) plants.

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FIGURE 5
Student responses to the question “How confident are you in your ability to use scientific literature and/or reports to guide research?” Pre-assessment results are in blue; post-assessment results are in red.

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FIGURE 6
Student responses to the question “How confident are you in your ability to develop theories (integrate and coordinate results from multiple studies)?” Pre-assessment results are in blue; post-assessment results are in red.

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FIGURE 7
Student responses to the statement “Please indicate the extent to which you agree with the following statement: I feel like I belong in the field of science.” Pre-assessment results are in blue; post-assessment results are in red.

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FIGURE 8
Student responses to the question “On a scale from 0 (least) to 10 (most), to what extent do you intend to pursue a science-related career?” Pre-assessment results are in blue; post-assessment results are in red.