Worms Rule- Investigating variation in isoform function

Anna Allen, Howard University

Location:

Abstract

Integrating research into undergraduate science courses has been a long-term goal of many institutions. Research-based laboratory courses provide students with authentic research experiences while also helping them develop their analytical thinking and problem solving skills. Through these type of courses, students begin to understand and apply many fundamental concepts in biology while also contributing to the scientific field. To provide a research experience consisting of many common laboratory skills and the current buzz technique of CRISPR/Cas9 endogenous genome editing, we designed a one-semester research experience for undergraduates. By the end of a single semester, students enrolled in our upper level biology elective course successfully edited the genome of the nematode Caenorhabditis elegans (C. elegans). Throughout this course, students were exposed to molecular biology techniques (PCR, gel electrophoresis), imaging techniques (confocal microscopy), and CRISPR/Cas9 concept and techniques in C. elegans. Ultimately, the goal of this course was to provide students with a meaningful undergraduate research experience while generating reagents (namely C. elegans strains) that assist the instructor's personal research objectives.

Research Goals

  1. Develop reagents (fluorescently tagged or mutant nematode strains) that will enable the students to study a gene of interest.
  2. Utilizing the reagents developed in Goal 1 to study either the spatiotemporal expression or the mutant phenotype of the C. elegans gene.

Student Goals related to Research Goal 1

  1. Develop basic molecular biology and C. elegans research skills
  2. Use information from primary literature to develop and defend the design of the CRISPR/Cas9 experiment
  3. Use knowledge of CRISPR/Cas9 to generate novel endogenously edited C. elegans strains

Student Goals related to Research Goal 2

  1. Analyze expression data from generated CRISPR reagents using live imaging microscopy
  2. Determine and explain any phenotypes of the CRISPR generated strains; make and defend the conclusions drawn from the data
  3. Communicate about research progress orally and in writing

Context

This semester-long CURE is intended to be offered for 16-20 students in an upper-level developmental biology course. The students should have a background in general biology and genetics. Ideally this course includes a laboratory that meets twice per week for 2-3 hours each. Since the laboratory utilizes the model organism C. elegans during certain aspects of the research, students would have to devote daily time in order to maintain their organisms and isolate transgenic animals. This research could involve live imaging microscopy and if that module is utilized the researchers will need access to an appropriate microscope.

Target Audience: Upper-level biology elective
CURE Duration: One-semester

CURE Design

Various isoforms are known to play unique spatial and temporal function during organismal development. This CURE is designed to investigate the differing functions of C. elegans genes with multiple splice isoforms. Through CRISPR/Cas9 endogenous genome editing, the student researchers will generate either fluorescently tagged specific isoforms or isoform mutants of the gene of interest. Students will be involved in planning, generating, and characterizing the CRISPR generated lines. Ultimately, the gene of interest can be altered depending on the principle investigator's individual laboratory's research interests.

Core Competencies: scientific research skills
Nature of Research: C. elegans, molecular biology

Tasks that Align Student and Research Goals

Research Goals →

Student Goals ↓

Research Goal 1: Develop reagents (fluorescently tagged or mutant nematode strains) that will enable the students to study a gene of interest.

Student Goal 1: Develop basic molecular biology and C. elegans research skills

  • Summarize current, general knowledge about C. elegans based on provided Genetics PRIMER article
  • Use microscopes to view, identify and move ("pick") C. elegans
  • Learn pipetting skills
  • Use Wormbase.org to investigate the gene of interest and find specific features (see WormBase Protocol included in main instructional material PDF)
  • Use PubMed or Google Scholar to find at least 5 papers published within the past five years on the study of the gene of interest in C. elegans
  • Create an annotated bibliography of at least 5 primary lit papers most relevant to our research goal
  • Explain the process of PCR and the necessary items to obtain a PCR fragment
  • Gain familiarity with free sequence analysis software, like ApE and NCBI Blast, to analyze genomic sequences
  • Design primers for a PCR reaction of your gene of interest
  • Plasmid Mini-prep mastery - Perform and analyze a standard PCR experiment (including gel electrophoresis)
  • Complete a PCR purification protocol



Student Goal 2: Use information from primary literature to develop and defend the design of the CRISPR/Cas9 experiment
  • Summarize current, general knowledge about CRISPR/Cas9 based on 3 provided reviews/videos
  • Describe a standard CRISPR experiment in C. elegans
  • Outline a timeline for student's experiment, including how and when animals will be grown, injections will take place, and data will be collected
  • Identification of PAM sites, design of crRNAs and repair oligonucleotides for gene of interest.
  • Defend your specific crRNA choice for your experiment to peers an instructor
  • Gather and summarize feedback from others (peers, instructor) about proposed experimental decisions



Student Goal 3: Use knowledge of CRISPR/Cas9 to generate novel endogenously edited C. elegans strains
  • Briefly summarize the current status of your experiment
  • Synthesize the appropriate repair template utilizing PCR.
  • Perform CRISPR injections following specified co-CRISPR protocol (injections typically done by instructional staff)
  • Identify successfully modified broods within the first generation after injection
  • Screen potential positive lines via PCR to verify edits at your gene of interest
  • Establish at least two independent edits (derived from different P0s) for each experiment.
  • Write (or present) brief progress reports, including descriptions of any issues or problems and how they were addressed / are being addressed




Research Goals →
Student Goals ↓
Research Goal 2: Utilizing the reagents developed in Goal 1 to study either the spatiotemporal expression or the mutant phenotype of the C. elegans gene.

Student Goal 1: Analyze expression data from generated CRISPR reagents using live imaging microscopy
  • Summarize current, general knowledge about live imaging microscopy based on provided resource articles
  • Learn microscopy skills on spinning disk confocal microscope
  • Practice taking images of previously generated fluorescent line
  • Generate multiple high quality images of fluorescently tagged CRISPR line



Student Goal 2: Determine and explain any phenotypes of the CRISPR generated strains; make and defend the conclusions drawn from the data
  • Analyze phenotypes of mutant strains.
  • Conduct fertility assay (or assay of choice) on mutant generated strains.



Student Goal 3: Communicate about research progress orally and in writing
  • Prepare and present periodic group / class meetings about the research
  • Write brief progress reports, including descriptions of any issues or problems and how they were addressed / are being addressed
  • Prepare and present a poster about the research to audiences outside the group / class (University Research Day)
  • Contribute to writing a paper about the research (e.g., draft methods and results)


Instructional Materials

A timeline and basic initial instructional material can be found combined in the following file: Worms Rule Timeline & Appendix (Microsoft Word 2007 (.docx) 932kB Feb10 18)

For a complete protocol on conducting homology-directed genome editing in C. elegans, implementors are directed to follow the protocol included in the publication: "High Efficiency, Homology-Directed Genome Editing in Caenhorhabditis elegans Using CRISPR-CAS9 Ribonucleoprotein Complexes" by Paix et al., Genetics 2015. This article contains a detailed CRISPR-HDF editing protocol as Supplemental File S1.

Additional basic C. elegans protocols can be found on www.wormbook.org

Assessment

For this CURE, I have assessed students through a mid-semester lab meeting presentation to the entire course, an end-of-semester lab meeting presentation to the entire course, and through generation of a written scientific paper detailing their results. The scientific paper is in the format of a short journal article complete with an abstract, introduction, materials & methods, results, discussion, and reference section.

The Participation Rubric that I use to grade other classmates during the lab meeting presentations can be found here: Participation Rubric (Acrobat (PDF) 62kB Feb10 18)

The Presentation Rubric that I use to grade students on their lab meeting presentations can be found here:Presentation Rubric (Acrobat (PDF) 63kB Feb10 18)

Instructional Staffing

Assisting students with the CURE work will be both peer mentors who have previously taken the course (UGAs) and graduate teaching assistants (GTAs). UGAs will become familiar with the CURE due to their previous participation in the research project. Their specific roles will be to assist with the preparation work, and assist students with training in the basic techniques utilized within the research project. The graduate teaching assistant will be the driving force behind instructing, training and advising the undergraduate researchers.

Author Experience

Anna Allen, Howard University

Advice for Implementation

The Author has taught using this CURE in two different semesters. The first semester, the class was filled with extremely research motivated undergraduates at both the junior and senior level, and the Author had both herself and a graduate student teaching assistant helping. The second iteration, consisted of a class comprised solely of seniors and while half the class was taking the course because of interest the other half was taking the course because of the convenient time offering. Additionally, the second time there was no graduate student teaching assistant and the Author was solely responsible for all instructional and laboratory aspects. The first iteration was much more successful when students were interested and willing to come into the laboratory even during non-class times.

This CURE is possible to complete in a semester, however, it does require students to do aspects of the research outside of the set classroom laboratory time. The benefit of having a Teaching Assistant is that they can help when the students may not be able to come into the lab. Finally, the Author has found that having students work in teams seemed to be beneficial because then the students could help each other cover the research time required outside of the set laboratory time.

For this CURE, it is necessary to have access to a microinjection system in order to generate the endogenously edited C. elegans strains. The most challenging aspect of the CURE is the physical injections of the nematodes, and for that an experienced injector (the PI or the Teaching Assistant) was needed. Cost wise, there are the costs of ordering the required oligonucleotides, sgRNAs and repair ssODNs. As this CURE can be beneficial to the instructor's own research project, some of these costs can be defrayed through using research funds.

Finally, please do not hesitate to contact the Author (Anna Allen, anna.allen@howard.edu) with any questions if you are considering implementing this particular CURE.

Iteration

The students are actively involved in trouble-shooting in many aspects of this CURE. Computationally, they spend time trying to identify the correct guide RNA and primer pairs to screen for successfully edited strains. The students can also conduct multiple PCRs (or a single gradient PCR) to identify the optimal annealing temperature for their screening primer pairs. There are many opportunities built in for students to learn how to pick and move C. elegans animals. This allows them to "fail" in maintaining their animals prior to having to be careful and gentle with the potentially edited animals.

Using CURE Data

Currently the data from this CURE has not been published, however students in both the first and second iteration have generated CRISPR edited C. elegans strains that are being utilized in the Author's research laboratory.

Resources

Articles
1. Introduction to Fluorescence Microscopy
3. Introductory Confocal Concepts
4. Ettinger A, Wittmann T. 2014. Quantitative Imaging in Cell Biology, Fluorescence live cell imaging, 1st ed. Academic Press.
5. Oreapoulos J, Berman R, Browne, M. 2014. Quantitative Imaging in Cell Biology, Spinning-disk confocal microscopy: present technology and future trends, 1st ed. Academic Press.
6. Corsi AK, Wightman B, Chalfie M. 2015. A Transparent window into biology: A primer on Caenorhabditis elegans. WormBook.
7. Dickinson DJ, Goldstein B. 2016. CRISPR-based methods for Caenorhabditis elegans genome engineering. Genetics 202:885–901. doi: 10.1534/genetics.115.182162.
8. Paix A, Folkmann A, Rasoloson D, Seydoux G. High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9. Genetics 201(1):47-54. DOI:10.1534/genetics.115.179382



Videos on CRISPR/Cas9 to Watch (in order)

1. Carl Zimmer explains the CRISPR DNA editing system in 90 seconds
2. Genome Editing with CRISPR-Cas9.
3. Genome Engineering with CRISPR-Cas9.