How to genetically engineer zebrafish
Summary by Will Anderson: Rafferty, S. A. & Quinn, T. A. A beginner's guide to understanding and implementing the genetic modification of zebrafish. Prog Biophys Mol Biol 138, 3-19, doi:10.1016/j.pbiomolbio.2018.07.005 (2018).
Image credit: Ciencias Españolas, Wikimedia Commons
Zebrafish are a popular vertebrate animal model for several reasons, including their genetic similarity to humans and their amenability to genetic manipulation. Techniques commonly performed in the zebrafish, such as mutagenic screens and various methods of insertion of foreign DNA, can reveal previously unknown genes and explore the function of characterized genes. Here, we discuss a review by Rafferty and Quinn that summarizes contemporary genetic modification and transgenesis in zebrafish1.
Screens to identify novel relationships between genes and phenotypes fall into two categories: forward and reverse genetic approaches. In forward genetic screens, investigators induce random mutations, observe resulting phenotype changes, and identify the causative mutation. In reverse genetic screens, researchers perform targeted gene knockdowns, then investigate any resulting phenotypic changes.
Several strategies to randomly disrupt gene activity and study resulting phenotypic changes have been used in zebrafish, such as ENU mutagenesis screens. In contrast, tools such as retroviral vectors and transposons are used to randomly insert known DNA sequences into the zebrafish genome. Retroviral vectors are useful because they can cause multiple heritable alterations in a single fish, while transposons can introduce a reporter sequence that helps identify expression changes1. In both methods, inserted sequences may disrupt genes and cause observable phenotypes, and the knowledge of the disrupting sequence provides a means to identify the perturbed locus.
In contrast to random genome disruption, the TALENs and CRISPR/Cas9 systems are popular targeted approaches to disrupt specific loci by inducing double strand breaks (DSBs)2. In the TALENs system, TALE proteins that can be designed to target specific DNA sequences are introduced to the single-cell zebrafish embryo and induce DSBs. In the CRISPR/Cas9 strategy, Cas9, a bacterial nuclease, is instructed by a “guide RNA” (gRNA) designed by an investigator to induce DSBs at targeted locations. These methods are useful not only for their ability to disrupt targeted DNA regions, but because when performed with the inclusion of exogenous DNA, they can be used to insert designed sequences into the zebrafish genome, creating transgenics. Transgenics can be useful in visualizing expression of genes via reporter proteins, altering existing gene function, and knocking in exogenous genes.
Methods such as “CRE/LOX”, the “Gal4/UAS” system, and optogenetic systems allow for conditional gene expression, improving the ability to determine where gene expression is required. The CRE/LOX system cleaves and rearranges specific DNA sequences when expressed, allowing for the conditional removal of targeted DNA sequences within a cut region. The Gal4/UAS system uses the combinatory effect of Gal4 and UAS DNA sequences to activate cell type-specific expression. Optogenetic approaches use light-responsive proteins to manipulate endogenous systems via the application of light.
Together, this plethora of tools allows zebrafish investigators many opportunities to explore vertebrate biology. The random disruption of the genome and random insertion of DNA sequences can enhance the understanding of the zebrafish genome, and targeted transgenesis allows for the creation of new genetic lines and thus many angles of exploration into gene function. These approaches enhance an investigator’s ability to perform novel experiments and make discoveries1.
References
Rafferty, S. A. & Quinn, T. A. A beginner's guide to understanding and implementing the genetic modification of zebrafish. Prog Biophys Mol Biol 138, 3-19, doi:10.1016/j.pbiomolbio.2018.07.005 (2018).
Albadri, S., Del Bene, F. & Revenu, C. Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish. Methods 121-122, 77-85, doi:10.1016/j.ymeth.2017.03.005 (2017).