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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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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 GF2014A broadly implementable research course in phage discovery and genomics for first-year undergraduate studentsMBio51e010511310.1128/mBio.01051-13244967953950523 http://dx.doi.org/10.1128/mBio.01051-13
9. Riordan SM, Heruth DP, Zhang LQ, Ye SQ2015Application of CRISPR/Cas9 for biomedical discoveriesCell Biosci53310.1186/s13578-015-0027-9261372164487574 http://dx.doi.org/10.1186/s13578-015-0027-9
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2017-01-29
2018-04-25

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
Download as Powerpoint
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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