Biology 423L: Developmental Biology Lab 

Genetics students are invited to enroll in Biology 423L spring semester.  The semester long lab class uses the CRISPR/Cas 9 system to perform gene editing in zebrafish.  Zebrafish are great model organisms in which to study vertebrate development with their low cost of culture and transparent bodies.  The zebrafish genome has been sequenced and is publicly available. The goal of the project is to make zebrafish mutants using the CRISPR/Cas 9 system which allows investigators to initiate double strand breaks in a specific DNA sequence.  When these breaks are resolved, a gene may be deleted or otherwise rendered non-functional.

Students choose their gene of interest from a list of genes expected to be involved in either vascular development or the formation of cancer or both.  The first part of the project involves determining what is already known about the gene from the research literature. Next comes an exercise in bioinformatics: annotating the gene (finding introns and exons) and designing a pair of oligonucleotides to target unique sequences in the first and last exons of the gene. Double stranded DNA are then synthesized, purified, and transcribed.  Synthesis of both dsDNA and RNA transcripts (guide RNAs) involves careful preparation of reactions and gel electrophoresis to check on products. 

Guide RNAs and Cas9 are then injected into Fli1-egfp transgenic embryos which can express green fluorescent protein; embryos were incubated and scanned for phenotypes over a five day period.

What's Next?

Some students are using this experience to jump into longer term research in Dr. Essner's lab and other labs on campus.