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First Year Course-Based Undergraduate Research Experience (CURE) Using the CRISPR/Cas9 Genome Engineering Technology in Zebrafish

    Authors: Jay M. Bhatt1, Anil Kumar Challa2,*
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    Affiliations: 1: Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294; 2: Department of Genetics, Transgenic & Genetically Engineered Models (TGEMs) Core Facility, University of Alabama at Birmingham, Birmingham, AL 35294
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    Source: J. Microbiol. Biol. Educ. January 2017 vol. 19 no. 1 doi:10.1128/jmbe.v19i1.1245
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    Abstract:

    Genetic analysis in model systems can provide a rich context for conceptual understanding of gene structure, regulation, and function. With an intent to create a rich learning experience in molecular genetics, we developed a semester-long course-based undergraduate research experience (CURE) using the CRISPR-Cas9 gene editing system to disrupt specific genes in the zebrafish. The course was offered to freshman students; nine students worked in four groups (two to three members per group) to design, synthesize, and test the nuclease activity of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/sgRNAs for targeted disruption of specific genes in the zebrafish. Each group worked with a gene with an already known mutant phenotype that can be visually scored and a gene that had not been studied in zebrafish previously. Embedded in the course were a series of workshop-styled units or tutorials, including tours to core facilities. The focus was on introducing and developing skills that could be accommodated within the span of a semester. Each group successfully cloned at least one plasmid-encoding CRISPR/sgRNA template, visually analyzed injected embryos, and performed genotyping assays to detect CRISPR-Cas9 activity. In-class discussions, a final end-of-semester written test, and group oral presentations were assessed for an understanding of the CRISPR-Cas9 system, application of the CRISPR-Cas9 system as a gene manipulation tool, and experimental methods used to create plasmid vectors and synthesize sgRNA. In addition, poster presentations were evaluated by faculty, graduate students, and senior undergraduate students at a University research exposition. Self-reflections in the form of group conversations were video recorded. All students (9/9) distinctly showed learning gains after completing the activity, but the extent of the gains was variable, as seen from results of a written test and poster presentation assessment. Qualitative analysis of evaluations and self-reporting data indicated several gains, suggesting that all students found many aspects of the CURE valuable and gained project-specific (conceptual) and transferrable skills (science process and science identity).

References & Citations

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3. Elgin SC, Bangera G, Decatur SM, Dolan EL, Guertin L, Newstetter WC, San Juan EF, Smith MA, Weaver GC, Wessler SR, Brenner KA, Labov JB 2016 Insights from a convocation: integrating discovery-based research into the undergraduate curriculum CBE Life Sci Educ 15 2 10.1187/cbe.16-03-0118 4909350 http://dx.doi.org/10.1187/cbe.16-03-0118
4. Laursen S, Hunter A-B, Seymour E, Thiry H, Melton G 2010 Undergraduate research in the sciences: engaging students in real science John Wiley & Sons San Francisco, CA
5. Lopatto D, Tobias S 2010 Science in solution: the impact of undergraduate research on student learning Council on Undergraduate Research Washington DC
6. 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
7. Rodenbusch SE, Hernandez PR, Simmons SL, Dolan EL 2016 Early engagement in course-based research increases graduation rates and completion of science, engineering, and mathematics degrees CBE Life Sci Educ 15 2 10.1187/cbe.16-03-0117 27252296 4909342 http://dx.doi.org/10.1187/cbe.16-03-0117
8. Jordan TC, Burnett SH, Carson S, Caruso SM, Clase K, DeJong RJ, Dennehy JJ, Denver DR, Dunbar D, Elgin SCR, Findley AM, Gissendanner CR, Golebiewska UP, Guild N, Hartzog GA, Grillo WH, Hollowell GP, Hughes LE, Johnson A, King RA, Lewis LO, Li W, Rosenzweig F, Rubin MR, Saha MS, Sandoz J, Shaffer CD, Taylor B, Temple L, Vazquez E, Ware VC, Barker LP, Bradley KW, Jacobs-Sera D, Pope WH, Russell DA, Cresawn SG, Lopatto D, Bailey CP, Hatfull GF 2014 A broadly implementable research course in phage discovery and genomics for first-year undergraduate students MBio 5 1 e01051 13 10.1128/mBio.01051-13 24496795 3950523 http://dx.doi.org/10.1128/mBio.01051-13
9. Riordan SM, Heruth DP, Zhang LQ, Ye SQ 2015 Application of CRISPR/Cas9 for biomedical discoveries Cell Biosci 5 33 10.1186/s13578-015-0027-9 26137216 4487574 http://dx.doi.org/10.1186/s13578-015-0027-9
10. Anorve-Andress K, Arcand AL, Borg BR, Brown JL, Chartrand CA, Frank ML, Jansen JN, Joyce MJ, Joyce MT, Kinney JA, Kruggel SL, Lecy AD, Ma P, Malecha KM, Melgaard K, Miller PL, Nelson KK, Nieto Robles M, Perosino TR, Peterson JM, Rollins AD, Scherkenbach WL, Smith AL, Sodergren KA, Stiller JJ, Wehber KR, Liang JO 2016 Variation in spot and stripe patterns in original and regenerated zebrafish caudal fins Zebrafish 13 4 256 265 10.1089/zeb.2015.1192 27096743 http://dx.doi.org/10.1089/zeb.2015.1192
11. Felzien LK 2016 Integration of a zebrafish research project into a molecular biology course to support critical thinking and course content goals Biochem Mol Biol Educ 44 6 565 573 10.1002/bmb.20983 27229632 http://dx.doi.org/10.1002/bmb.20983
12. Marra MH, Tobias ZJ, Cohen HR, Glover G, Weissman TA 2015 In vivo time-lapse imaging in the zebrafish lateral line: a flexible, open-ended research project for an undergraduate neurobiology laboratory course J Undergrad Neurosci Educ 13 3 A215 A224 26240532 4521740
13. Sarmah S, Chism GW 3rd, Vaughan MA, Muralidharan P, Marrs JA, Marrs KA 2016 Using zebrafish to implement a course-based undergraduate research experience to study teratogenesis in two biology laboratory courses Zebrafish 13 4 293 304 10.1089/zeb.2015.1107 26829498 http://dx.doi.org/10.1089/zeb.2015.1107
14. Roberts JR, Hagedorn E, Dillenburg P, Patrick M, Herman T 2005 Physical models enhance molecular three-dimensional literacy in an introductory biochemistry course Biochem Mol Biol Educ 33 2 105 110 10.1002/bmb.2005.494033022426 21638554 http://dx.doi.org/10.1002/bmb.2005.494033022426
15. Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F 2013 DNA targeting specificity of RNA-guided Cas9 nucleases Nat Biotechnol 31 9 827 832 10.1038/nbt.2647 23873081 3969858 http://dx.doi.org/10.1038/nbt.2647
16. Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK 2013 Efficient genome editing in zebrafish using a CRISPR-Cas system Nat Biotechnol 31 3 227 229 10.1038/nbt.2501 23360964 3686313 http://dx.doi.org/10.1038/nbt.2501
17. Challa AK, Boitet ER, Turner AN, Johnson LW, Kennedy D, Downs ER, Hymel KM, Gross AK, Kesterson RA 2016 Novel hypomorphic alleles of the mouse tyrosinase gene induced by CRISPR-Cas9 nucleases cause non-albino pigmentation phenotypes PLoS ONE 11 5 e0155812 10.1371/journal.pone.0155812 27224051 4880214 http://dx.doi.org/10.1371/journal.pone.0155812
18. Hanauer DI, Frederick J, Fotinakes B, Strobel SA 2012 Linguistic analysis of project ownership for undergraduate research experiences CBE Life Sci Educ 11 4 378 385 10.1187/cbe.12-04-0043 23222833 3516793 http://dx.doi.org/10.1187/cbe.12-04-0043
19. Gagnon JA, Valen E, Thyme SB, Huang P, Akhmetova L, Pauli A, Montague TG, Zimmerman S, Richter C, Schier AF 2014 Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs PLoS ONE 9 5 e98186 10.1371/journal.pone.0098186 24873830 4038517 http://dx.doi.org/10.1371/journal.pone.0098186

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2017-01-29
2019-08-25

Abstract:

Genetic analysis in model systems can provide a rich context for conceptual understanding of gene structure, regulation, and function. With an intent to create a rich learning experience in molecular genetics, we developed a semester-long course-based undergraduate research experience (CURE) using the CRISPR-Cas9 gene editing system to disrupt specific genes in the zebrafish. The course was offered to freshman students; nine students worked in four groups (two to three members per group) to design, synthesize, and test the nuclease activity of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/sgRNAs for targeted disruption of specific genes in the zebrafish. Each group worked with a gene with an already known mutant phenotype that can be visually scored and a gene that had not been studied in zebrafish previously. Embedded in the course were a series of workshop-styled units or tutorials, including tours to core facilities. The focus was on introducing and developing skills that could be accommodated within the span of a semester. Each group successfully cloned at least one plasmid-encoding CRISPR/sgRNA template, visually analyzed injected embryos, and performed genotyping assays to detect CRISPR-Cas9 activity. In-class discussions, a final end-of-semester written test, and group oral presentations were assessed for an understanding of the CRISPR-Cas9 system, application of the CRISPR-Cas9 system as a gene manipulation tool, and experimental methods used to create plasmid vectors and synthesize sgRNA. In addition, poster presentations were evaluated by faculty, graduate students, and senior undergraduate students at a University research exposition. Self-reflections in the form of group conversations were video recorded. All students (9/9) distinctly showed learning gains after completing the activity, but the extent of the gains was variable, as seen from results of a written test and poster presentation assessment. Qualitative analysis of evaluations and self-reporting data indicated several gains, suggesting that all students found many aspects of the CURE valuable and gained project-specific (conceptual) and transferrable skills (science process and science identity).

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Figures

Image of FIGURE 1

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FIGURE 1

Course overview with modules, objectives, and outcomes. The orange boxes show the preparatory and core laboratory modules leading up to the poster presentations. The violet boxes show the modules that support the core laboratory modules.

Source: J. Microbiol. Biol. Educ. January 2017 vol. 19 no. 1 doi:10.1128/jmbe.v19i1.1245
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Image of FIGURE 2

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FIGURE 2

Workflow of the molecular biology lab module. Experiments included construction and linearization of a CRISPR/sgRNA template, transcription of sgRNA, injection of sgRNA with Cas9 protein, observation and analysis of development in injected embryos, and genotyping injected embryos by PCR to detect CRISPR-Cas9 nuclease activity. Numbers indicated in blue circles represent the week in which the activity/procedure was done. CRISPR = clustered regularly interspaced short palindromic repeats; PCR = polymerase chain reaction.

Source: J. Microbiol. Biol. Educ. January 2017 vol. 19 no. 1 doi:10.1128/jmbe.v19i1.1245
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Image of FIGURE 3

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FIGURE 3

Assessment of student learning from poster presentations at the undergraduate research exposition. Student presentations were assessed based on their demonstrated understanding in four areas. The x-axis represents the total number of poster evaluations.

Source: J. Microbiol. Biol. Educ. January 2017 vol. 19 no. 1 doi:10.1128/jmbe.v19i1.1245
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Image of FIGURE 4

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FIGURE 4

Post-course survey responses. Student responses (=7 students) on the value of activities performed during the course.

CRISPR = clustered regularly interspaced short palindromic repeats; PCR = polymerase chain reaction.

Source: J. Microbiol. Biol. Educ. January 2017 vol. 19 no. 1 doi:10.1128/jmbe.v19i1.1245
Download as Powerpoint

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