Major
Biological Sciences
Advisor
Irvine, Steven
Advisor Department
Biological Sciences
Date
5-2018
Keywords
Genetics; CRISPR; Mutation; Tyrosinase; Ciona intestinalis
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
Abstract
Genetic manipulation has come a long way in the past ten years alone. Scientists have had access to gene editing techniques for decades, but until recently these methods have proven to be expensive and unpredictable. However, thanks to the development of a new, more efficient genome editing strategy called CRISPR/Cas9, more aggressive progress can now be made in genetics research.
CRISPR is not a machine or a physical tool, but rather it is a system that involves introducing a protein into a cell, along with a DNA segment that will attract the protein to a desired location on the DNA. The Cas9 protein then induces a double stranded break at the location, silencing whichever gene is located there.
The major goal of my project was to demonstrate the successful implementation of CRISPR using embryos spawned from the model chordate Ciona intestinalis, also known as the sea squirt. We will use the system to inactivate the gene in Ciona embryos which encodes for the tyrosinase enzyme responsible for producing the pigment seen in the dark eye spots of Ciona embryos during the late tailbud stage. In mutated embryos, the tyrosinase gene becomes inactivated by the CRISPR system, causing the entire larvae to appear white under the microscope with no visible eye spots.
This experiment provides a proof of concept for the use of CRISPR/Cas9 in the Irvine lab. Ongoing experiments utilizing CRISPR aim to silence other genes involved in temperature response pathways. These tests will further our understanding of how projected increases in ocean temperatures will impact reproduction in Ciona populations, and potentially in other aquatic species as well.
Comments
The aim of my project was to demonstrate the successful implementation of a new tool for genome editing called CRISPR using embryos spawned from the model chordate Ciona intestinalis. The CRISPR system was developed in 2013 and it has since been proven to be a functionally much simpler strategy for gene manipulation compared to previous techniques. My goal was to conduct experiments which demonstrate the CRISPR system’s ability to silence a gene in Ciona intestinalis. I intended to use CRISPR to inactivate the TYR gene in Ciona embryos, a gene which encodes for the tyrosinase enzyme responsible for producing the pigment seen in the dark eye spots of Ciona embryos during the late tailbud stage. In the mutated embryos, the TYR gene is inactivated by the CRISPR system, causing the entire embryo to appear white under the microscope with no visible eye spots, without changing the other characteristics of the developing embryo.
The procedure to generate the construct to be electroporated into the Ciona embryos involves running polymerase chain reactions (PCRs) using four primers and two templates. Two sequence-specific guide-RNA primers target the TYR gene and anneal it to a sequence containing the primers for the U6 promoter and the guide-RNA scaffold. These components were amplified many times by PCR to produce many complete constructs. Once the Ciona become gravid during the warmer months, the chorions (outer membranes) of newly spawned Ciona embryos will be removed using a protease. After that, the CRISPR constructs along with vectors containing Cas9 protein-coding sequences will be inserted into the dechorionated embryos by electroporation. This technique uses a small pulse of electricity to open the pores of cell membranes and allow DNA to be introduced into the cells. The construct will then bind with DNA in the Ciona embryos, and Cas9 proteins will recognize that sequence and target it for destruction, thereby inactivating the TYR gene.