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An Undergraduate Research Project Utilizing CRISPR-Cas9 Gene Editing Technology to Study Gene Function in

    Authors: Nicholas J. Ruppel1, Lauren E. Estell1, Robert I. Jackson2, Michael J. Wolyniak2
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    Affiliations: 1: Department of Biology, Randolph-Macon College, Ashland, VA 23005; 2: Department of Biology, Hampden-Sydney College, Hampden-Sydney, VA 23943
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    Source: J. Microbiol. Biol. Educ. June 2019 vol. 20 no. 2 doi:10.1128/jmbe.v20i2.1666
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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 class laboratories, emphasizing its use in plants. Our genetic manipulations were designed for , 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 . 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

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2. Adli M 2018 The CRISPR tool kit for genome editing and beyond Nat Commun 9 1911 10.1038/s41467-018-04252-2 http://dx.doi.org/10.1038/s41467-018-04252-2
3. Scheben A, Edwards D 2018 Towards a more predictable plant breeding pipeline with CRISPR/Cas-induced allelic series to optimize quantitative and qualitative traits Curr Opin Plant Biol 45 218 225 10.1016/j.pbi.2018.04.013 29752075 http://dx.doi.org/10.1016/j.pbi.2018.04.013
4. Biagioni A, Chillà A, Laurenzana A, Margheri F, Peppicelli S, Del Rosso M, Fibbi G 2017 Type II CRISPR/Cas9 approach in the oncological therapy J Exp Clin Cancer Res 36 80 10.1186/s13046-017-0550-0 28619109 5472952 http://dx.doi.org/10.1186/s13046-017-0550-0
5. Brokowski C, Adli M 2019 CRISPR ethics: moral considerations for applications of a powerful tool J Mol Biol 431 88 101 10.1016/j.jmb.2018.05.044 http://dx.doi.org/10.1016/j.jmb.2018.05.044
6. Thurtle-Schmidt DM, Lo T 2018 Molecular biology at the cutting edge: a review on CRISPR/CAS9 gene editing for undergraduates Biochem Mol Biol Educ 46 195 205 10.1002/bmb.21108 29381252 5901406 http://dx.doi.org/10.1002/bmb.21108
7. Bhatt JM, Challa AK 2017 First year course-based undergraduate research experience (CURE) using the CRISPR/Cas9 genome engineering technology in zebrafish J Microbiol Biol Educ 19 1 29904527 5969413
8. Auchincloss LC, Laursen SL, Branchaw JL, Eagan K, Graham M, Hanauer DI, Lawrie G, McLinn CM, Pelaez N, Rowland S, Towns M, Trautmann NM, Varma-Nelson P, Weston TJ, Dolan EL 2014 Assessment of course-based undergraduate research experiences: a meeting report CBE Life Sci Educ 13 1 29 40 10.1187/cbe.14-01-0004 24591501 3940459 http://dx.doi.org/10.1187/cbe.14-01-0004
9. Anderson HJE 2017 CRISPR in the undergraduate classroom: a CURE FASEB J 31 Suppl 1 589.6
10. Adame V, Chapapas H, Cisneros M, Deaton C, Deichmann S, Gadek C, Lovato TL, Chechenova MB, Guerin P, Cripps RM 2016 An undergraduate laboratory class using CRISPR/Cas9 technology to mutate Drosophila genes Biochem Mol Biol Educ 44 263 275 10.1002/bmb.20950 27009801 5377917 http://dx.doi.org/10.1002/bmb.20950
11. Ma X, Zhu Q, Chen Y, Liu YG 2016 CRISPR/Cas9 platforms for genome editing in plants: developments and applications Mol Plant 9 961 974 10.1016/j.molp.2016.04.009 27108381 http://dx.doi.org/10.1016/j.molp.2016.04.009
12. Molina I, Weber K, Alves Cursino, dos Santos DY, Ohlrogge J 2008 Transformation of a dwarf Arabidopsis mutant illustrates gibberellin hormone physiology and the function of a green revolution gene Biochem Mol Biol Educ 37 3 170 177 10.1002/bmb.20263 http://dx.doi.org/10.1002/bmb.20263
13. Estrada M, Woodcock A, Hernandez PR, Schultz P 2011 Toward a model of social influence that explains minority student integration into the scientific community J Educ Psychol 103 206 222 10.1037/a0020743 21552374 3087606 http://dx.doi.org/10.1037/a0020743
14. Corwin LA, Runyon C, Robinson A, Dolan EL 2015 The laboratory course assessment survey: a tool to measure three dimensions of research course-design CBE Life Sci Educ 14 ar37 10.1187/cbe.15-03-0073 http://dx.doi.org/10.1187/cbe.15-03-0073
15. Yang M, Sack FD 1995 The too many mouths and four lips mutations affect stomatal production in Arabidopsis Plant Cell 7 2227 2239 11536724 161075
16. Herman PL, Marks MD 1989 Trichome development in Arabidopsis thaliana II Isolation and complementation of the GLABROUS1 gene Plant Cell 1 1051 1055 10.2307/3869022 12359886 159842 http://dx.doi.org/10.2307/3869022
17. Di Laurenzio L, Wysocka-Diller J, Malamy JE, Pysh L, Helariutta Y, Freshour G, Hahn MG, Feldmann KA, Benfey PN 1996 The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root Cell 86 423 433 10.1016/S0092-8674(00)80115-4 8756724 http://dx.doi.org/10.1016/S0092-8674(00)80115-4
18. Koornneef M, Rolff E, Spruit CJP 1980 Genetic control of light-inhibited hypocotyl elongation in Arabidopsis thaliana (L) HEYNH Z Pflanzenphysiol 100 147 160 10.1016/S0044-328X(80)80208-X http://dx.doi.org/10.1016/S0044-328X(80)80208-X
19. Lee MM, Schiefelbein J 1999 WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning Cell 99 473 483 10.1016/S0092-8674(00)81536-6 10589676 http://dx.doi.org/10.1016/S0092-8674(00)81536-6
20. Caspar T, Pickard BG 1989 Gravitropism in a starchless mutant of Arabidopsis Planta 177 185 197 10.1007/BF00392807 24212341 http://dx.doi.org/10.1007/BF00392807
21. Reiser L, Subramaniam S, Li D, Huala E 2017 Using the Arabidopsis information resource (TAIR) to find information about Arabidopsis genes Curr Protoc Bioinformatics 60 10.1002/cpbi.36 29220077 http://dx.doi.org/10.1002/cpbi.36
22. Xie K, Zhang J, Yang Y 2014 Genome-wide prediction of highly specific guide RNA spacers for the CRISPR-Cas9 mediated genome editing in model plants and major crops Mol Plant 7 923 926 10.1093/mp/ssu009 24482433 http://dx.doi.org/10.1093/mp/ssu009
23. Weigel D, Glazebrook J 2006 Transformation of Agrobacterium using the freeze-thaw method CSH Protoc 2006 7 10.1101/pdb.prot4666 http://dx.doi.org/10.1101/pdb.prot4666
24. Clough SJ, Bent AF 1998 Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana Plant J 16 735 743 10.1046/j.1365-313x.1998.00343.x http://dx.doi.org/10.1046/j.1365-313x.1998.00343.x
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2019-06-28
2019-07-20

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 class laboratories, emphasizing its use in plants. Our genetic manipulations were designed for , 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 . 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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Figures

Image of FIGURE 1

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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 T transgenic plants were growing. CRISPR = clustered regularly interspaced short palindromic repeat.

Source: J. Microbiol. Biol. Educ. June 2019 vol. 20 no. 2 doi:10.1128/jmbe.v20i2.1666
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Image of FIGURE 2

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FIGURE 2

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

Source: J. Microbiol. Biol. Educ. June 2019 vol. 20 no. 2 doi:10.1128/jmbe.v20i2.1666
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Image of FIGURE 3

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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 -test, <0.01, =41). Bars represent standard error.

Source: J. Microbiol. Biol. Educ. June 2019 vol. 20 no. 2 doi:10.1128/jmbe.v20i2.1666
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Image of FIGURE 4

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FIGURE 4

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

Source: J. Microbiol. Biol. Educ. June 2019 vol. 20 no. 2 doi:10.1128/jmbe.v20i2.1666
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Image of FIGURE 5

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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.

Source: J. Microbiol. Biol. Educ. June 2019 vol. 20 no. 2 doi:10.1128/jmbe.v20i2.1666
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Image of FIGURE 6

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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.

Source: J. Microbiol. Biol. Educ. June 2019 vol. 20 no. 2 doi:10.1128/jmbe.v20i2.1666
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Image of FIGURE 7

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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.

Source: J. Microbiol. Biol. Educ. June 2019 vol. 20 no. 2 doi:10.1128/jmbe.v20i2.1666
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Image of FIGURE 8

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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.

Source: J. Microbiol. Biol. Educ. June 2019 vol. 20 no. 2 doi:10.1128/jmbe.v20i2.1666
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