Gene manipulation in zebrafish using the CRISPR-Cas9 system

Diane Tuckerdtucker@uab.eduUniversity of Alabama at Birmingham
Jay Bhattjaybhatt@uab.eduUniversity of Alabama at Birmingham

Project Location

AL
US
This CURE aims at introducing students to the the world of genome engineering using the CRISPR-Cas9 system. Students will learn basic to advanced concepts in molecular genetics in the context of genome engineering.

Student Audience

Introductory, Advanced

Scientific Domain

  • Bioinformatics/Computational biology
  • Developmental biology
  • Genetics/Genomics
  • Molecular and cellular biology

Nature of the Research

  • Wet lab/bench research
  • Informatics/computational research
  • Basic research
  • Database research

Core Concepts

  • Evolution: The diversity of life evolved over time by process of mutation, selection, and genetic change.
  • Structure and Function: Basic units of structure define the function of all living things.
  • Information Flow and Exchange: The growth and behavior of organisms are activated through the expression of genetic information in context.

Core Competencies

  • Applying the process of science
  • Communicating and collaborating
  • Understanding the relationship between science and society

Guiding Questions

  • What is a gene, and how are genes organized in the genome?
  • What are the different ways gene function and regulation be studied?
  • What is a mutation, and how can mutations help understand gene function?
  • How can model organisms help understand general principles of gene function?

Learning Objectives

  • Identify specific sequences in the zebrafish genome using ENSEMBL genome browser.
  • Design CRISPR/sgRNAs to target specific sequences using online tools
  • Prepare CRISPR/sgRNA by in vitro transcription
  • Assess nuclease activity of CRISPR/Cas9 by PCR-heteroduplex mobility assay
  • Present research question, methodology and results to science practitioners

Overview

The larger goal of the project is to introduce genome engineering as a tool to understand gene function using the zebrafish as a model system. Students learn to browse and analyze the zebrafish genome (using the ENSEMBL browser) to identify sequences that can be targeted by the CRISPR-Cas9 system. In this process, they also learn how to manage and annotate gene sequences using standalone and online tools (e.g. SnapGene and Benchling, respectively). This is followed by wet lab work to prepare CRISPR/sgRNA using in vitro synthesis, which will be injected into zebrafish embryos. Students visually observe and document the injected embryos for 3-4 days (post fertilization, dpf) for consistent, scorable phenotypes. Further, genomic DNA is isolated from these embryos (3-5 dpf) to assess nuclease activity of the injected CRISPR/sgRNAs using PCR and gel electrophoresis. Experimental validation of potentially useful CRISPR/sgRNAs is an important outcome of this project. Attributing function to uncharacterized or poorly understood gene sequences is also eminently possible.

Getting Started

A basic molecular biology lab is required to do the first and third (last) part of the project. Access to zebrafish embryos, and the ability to perform microinjection experiments is needed for the second part of the project.

Preparation

4-5 hours

Member Contact

Tucker, Diane dtucker@uab.edu University of Alabama at Birmingham
Bhatt, Jay jaybhatt@uab.edu University of Alabama at Birmingham

Class time

2-3 hours