The zebrafish has characteristics that make it an ideal model organism for studying genetic determinants that participate in development and disease. However, one major limitation to its widespread use is the absence of a technology that allows direct genomic manipulation of a desired target gene either to inactivate or alter its function. Recent studies in human cell lines, insects and plants indicate that site-specific mutations can be introduced using zinc finger nucleases (ZFNs). Using a combination of rational design and selection, we have successfully engineered ZFNs that promote targeted gene disruption in zebrafish that are transmissible through the germline. Using this technology it is now possible to generate targeted mutations throughout the zebrafish genome. In this grant proposal, we propose to optimize and extend the use of ZFNs in zebrafish. A major goal of the proposed studies will be to enable individual researchers to generate knockout or knock-in zebrafish lines in their own laboratories with modest technical effort. We will streamline the creation of site- specific zinc finger proteins the targeting component of ZFNs to simplify the process of making gene knockouts. We will further utilize and optimize ZFN technology for targeted homologous recombination with a DNA donor supplied in trans allowing targeted gene knock-ins. We will demonstrate the utility of this technology by making conditional alleles of genes involved in heart regeneration to allow the study of knockouts of required genes in the adult. We will also generate an allelic series of mutations in the flt4 receptor tyrosine kinase to better understand its role at multiple steps in vascular development. By providing an accessible technological platform for genetic manipulation to the zebrafish community, this study will ultimately enable the creation of a variety of gene knockouts or knock-ins that can be used in conjunction with powerful forward and reverse genetic screens in the zebrafish to study human disease models. Thus, the development and use of ZFN technology in the zebrafish will have far- reaching significance in virtually all areas of biomedical research.

Public Health Relevance

We are developing technology to allow the genome of the zebrafish to be engineered. This technology will allow researchers to construct models of human disease in this animal to understand its origin and develop better treatments. We will apply this technology in zebrafish to better understand the processes involved in heart regeneration and blood vessel formation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL093766-02
Application #
7664401
Study Section
Special Emphasis Panel (ZRG1-BDA-F (50))
Program Officer
Schramm, Charlene A
Project Start
2008-08-01
Project End
2013-05-31
Budget Start
2009-06-01
Budget End
2010-05-31
Support Year
2
Fiscal Year
2009
Total Cost
$415,564
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Genetics
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Bolukbasi, Mehmet Fatih; Liu, Pengpeng; Luk, Kevin et al. (2018) Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing. Nat Commun 9:4856
Zhu, Lihua Julie; Lawrence, Michael; Gupta, Ankit et al. (2017) GUIDEseq: a bioconductor package to analyze GUIDE-Seq datasets for CRISPR-Cas nucleases. BMC Genomics 18:379
Bolukbasi, Mehmet Fatih; Gupta, Ankit; Wolfe, Scot A (2016) Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery. Nat Methods 13:41-50
Shin, Masahiro; Male, Ira; Beane, Timothy J et al. (2016) Vegfc acts through ERK to induce sprouting and differentiation of trunk lymphatic progenitors. Development 143:3785-3795
Lawson, Nathan D (2016) Reverse Genetics in Zebrafish: Mutants, Morphants, and Moving Forward. Trends Cell Biol 26:77-79
Kok, F O; Shin, M; Ni, C-W et al. (2015) Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish. Dev Cell 32:97-108
Bolukbasi, Mehmet Fatih; Gupta, Ankit; Oikemus, Sarah et al. (2015) DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nat Methods 12:1150-6
Kok, Fatma O; Lawson, Nathan D (2015) A platform for reverse genetics in endothelial cells. Circ Res 117:107-8
Weicksel, Steven E; Gupta, Ankit; Zannino, Denise A et al. (2014) Targeted germ line disruptions reveal general and species-specific roles for paralog group 1 hox genes in zebrafish. BMC Dev Biol 14:25
Gupta, Ankit; Hall, Victoria L; Kok, Fatma O et al. (2013) Targeted chromosomal deletions and inversions in zebrafish. Genome Res 23:1008-17

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