Using the CRISPR-Cas9 genome engineering technology to understand gene function in the zebrafish

Anil Kumar Challa, University of Alabama at Birmingham
Dr. Diane Tucker, University of Alabama at Birmingham
Dr. Jay Bhatt, Creighton University
Dr. Ashley Turner, University of Alabama at Birmingham
Location: Alabama

Abstract

The CURE with CRISPR aims to introduce experimental research to students using cutting-edge genome engineering technologies driven by RNA-guided programmable nucleases. Using a combination of bioinformatics and lab bench ('wet lab') tools, students design, synthesize and analyze CRISPR reagents that can effectively target specific sites in the genome. We use the zebrafish as a model system to understand gene function. Ancillary activities like tours to the zebrafish and sequencing facilities are also included to expose students to various aspects that are integral to a research program.

Student Goals

  1. To experience the scientific process through explorations in bioinformatics and experimental molecular biology/genetics - students browse the zebrafish genome, analyze various aspects of a specific gene, design CRISPR targets for disruption of the gene, synthesize guide RNAs and validate the ability of the guide RNAs to perform site specific cleavage by the Cas9 protein.
  2. To generate, analyze and interpret experimental results
  3. To communicate scientific/research findings effectively

Research Goals

  1. Identify and validate CRISPR targets (in vitro and in vivo)
  2. Score potential phenotypes arising from mutating specific sequences in the zebrafish system
  3. Perform sequence analysis of mutant alleles

Context

The Science and Technology Honors Program at the University of Alabama at Birmingham has a two-semester sequence for incoming freshman. In the first (STH199) students (around 60) learn to read, analyze and interpret a scientific paper. The second course (STH201) introduces students (in smaller groups of 10-24) to research approaches in which they can choose from a few themes. Typically, these courses focus on building technical skills in specific scientific disciplines. This CURE was developed as part of the Molecular Genetics section of STH201. The CURE is designed to complete in one semester (Spring), although additional work in the following semester (Summer) has been voluntarily taken up by students. Students come in with a basic knowledge of biological sciences, especially genetics.

Students work in groups of 3-4, with each group focusing on a specific research outcome. Research outcomes of each group fit with a larger outcome for the entire class.

While the CURE was offered to first year students, the overall approach can be effectively used for upperclass students as well.

Target Audience: First year (for an overview of concepts in molecular genetics and a generic research experience), sophomore-senior year (microbiology, molecular genetics/genomics, developmental biology)
CURE Duration:A full term Full term. Modules can be done in 3-4 week blocks.

CURE Design

With growing insights into the workings of the genome, the definition of a gene is constantly evolving. There are several unknowns about the genome and genome sequences. Fortuitously, we now have technologies (CRISPR-Cas9) that enable us to precisely target specific sequences in the genome and query their function.

In the first part, students learn bioinformatics tools to explore and query the zebrafish genome. With guidance, they choose genes whose functions are poorly understood or not known and analyze those gene sequences. They develop basic biological insights into the gene structure and function.

In the second part, students learn and use experimental tools to generate CRISPR reagents to target specific gene/sequences. They perform experiments to validate the functionality of the reagents they created using in vitro systems and in vivo system (with the instructor's help).

Each group (3-4 students) is assigned a specific research outcome (typically a gene)
Each student in the group has to identify a specific target in that gene, based on biological insights
The data (both positive and negative) generated is analyzed and interpreted by each student through individual assignments, and each group, and the entire class through classroom discussions and peer review.

In the last part of the CURE, students are re-assigned to new 'poster' groups where they work together to write and submit a scientific abstract, and subsequently, prepare and present a poster at a student research exposition.

Core Competencies:Analyzing and interpreting data, Asking questions (for science) and defining problems (for engineering), Constructing explanations (for science) and designing solutions (for engineering), Developing and using models, Planning and carrying out investigations, Using mathematics and computational thinking
Nature of Research: Bioinformatics and 'wet lab'

Tasks that Align Student and Research Goals

Research Goals →
Student Goals ↓
Research Goal 1: Identify and validate CRISPR targets
Research Goal 2: Score potential phenotypes arising from mutating specific sequences in the zebrafish system


Student Goal 1: To experience the scientific process through explorations in bioinformatics and experimental molecular biology

Students use the ENSEMBL zebrafish genome browser to analyze gene sequences and Benchling tools to identify sites for CRISPR targeting followed by in vitro synthesis and validation of guide RNAs

Students analyze zebrafish embryos microinjected with in vitro validated guide RNAs using simple bright field microscopy



Student Goal 2: To generate, analyze and interpret experimental results

Students prepare specific guide RNAs by in vitro transcription and use them in an in vitro nuclease assay, analyze the nuclease assay products using gel electrophoresis, and infer the quality and efficiency of the guide RNAs

Students observe microinjected zebrafish embryos under a microscope and score for the presence of any phenotypic abnormalities in comparison to uninjected wild-type embryos, infer developmental defects, and correlate the defects to the potential roles of the specific genes under investigation



Student Goal 3: To communicate scientific/research findings effectively

Student teams prepare abstracts and posters to present at the undergraduate research exposition judged by the university scientific community

Student teams prepare abstracts and posters to present at the undergraduate research exposition judged by the university scientific community


Instructional Materials



Assessment

Formative assessments:
Team presentations followed by class discussions and peer assessments at important junctures in the experimental workflow.
Short, open-ended, feedback at the end of each week to reflect on the work done (in the past week) and work planned (for next week).

Summative assessments:
In class final exam to test comprehension of course content (conceptual and technical).
Abstract writing/submission, poster preparation and presentation at the University Student Research Exposition (UABExpo).


Instructional Staffing

The CURE was offered by the faculty/research expert assisted by postdoctoral fellows and undergraduate TAs who are CURE alumni.

Author Experience

Anil Kumar Challa, University of Alabama at Birmingham

Authentic research is an effective way to engage undergraduate students in the process of science and scientific insights. It also provides a rich context into which students can begin to integrate the knowledge they acquire. A CURE provides many valuable opportunities to engage a group of students with authentic research.


Read Story »

Advice for Implementation

There are multiple modules embedded in the CURE with CRISPR that can be easily implemented based on the needs and available classroom/laboratory resources.

Bioinformatics module: Using genome browsers (e.g. ENSEMBL) and online CRISPR design tools (e.g. Benchling) students can analyze gene sequences to assess what sites would be particularly useful for gene disruptions or editing. In the context of 'reverse genetics' studies where genes are disrupted to understand their functions, targeting early segments of the gene sequence to achieve early truncations or targeting regions/domains important for specific biological structure and/or function can be one objective. In case of gene editing disease mutations, the focus can be on identifying target sequences and strategies for correction of mutant sequences.

Nucleic acid biochemistry and molecular biology module: Using DNA and RNA polymerases, CRISPR/sgRNA synthesis can be done to introduce concepts in replication and transcription. Furthermore, the functional activity of the sgRNA (in complex with Cas9 protein) can be assessed in an in vitro nuclease assay using PCR-derived genomic fragment. This module aligns well with the flow of genetic information.

Molecular Genetics module: The in vitro validated sgRNA can be delivered into embryos (either in-house or as part of a collaboration) and studied for effects of gene disruption. Students can use simple, brightfield microscopy to observe any phenotypic abnormalities that may arise due to gene disruption, and/or PCR-based approaches to test whether the targeted gene/sequence has been modified.


Resources




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