Project Details
Description
The goals of this application are to apply tools and refine methodologies for
genome engineering in zebrafish that allow the creation of mutagenic and conditional
alleles that provide spatial and temporal control of gene expression. In doing so we will be able
to define genes that aid in the development and implementation of methodology to improve our
understanding of genetic manipulations that can promote or restore health. The ability to make
site-specific, untagged mutant alleles in zebrafish and other models has been greatly advanced
by custom nucleases that include TALENs and CRISPR/Cas9 systems. These systems operate
on the same principle: they are designed to bind to specific sequences in the genome and
create a double strand break. During the first granting cycle, we have leveraged this ability to
develop reliable methods for site-specific gene integration directed by short regions of
homologous sequence. In our renewal application, we utilize the tools, vectors and
methodologies generated to create both revertible alleles and Cre/Cre-ER lines for the zebrafish
community in Aim 1. In Aim 2, we will examine methodologies to improve site-specific
integration and the role of microhomology at CRISPR/Cas9 cut sites and new ways to present
the vector with microhomology to the genomic cut site. In Aim 3, we will continue to develop and
implement targeted integration resources by hosting workshops and onsite visits at both the
Mayo Clinic and Iowa State University. The tools, techniques and zebrafish lines produced will
have direct implications for providing precise gene editing techniques and critical lines to
examine genes with the potential to restore human health. We anticipate these methodologies
will enhance the efficiency of gene editing and will be readily adaptable for use in other model
organisms and large animals. In our opinion, this will have important implications for modeling
human disease in animal systems through the ability to utilize conditional alleles. The methods,
cargos and zebrafish lines are designed to significantly enhance identification of genes that
promote health through leveraging genome modification to produce conditional and revertible
alleles and alleles that mirror mutations in humans.
Status | Finished |
---|---|
Effective start/end date | 6/1/16 → 2/29/24 |
Funding
- NIH Office of the Director: $768,087.00
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