Presentation Title

Using CRISPR Based Genome Editing to Create an Autistic Zebrafish Model

Location

Nessmith-Lane Atrium

Session Format

Poster Presentation

Research Area Topic:

Natural & Physical Sciences - Biology

Abstract

In 2015, Autism Spectrum Disorder (ASD) was estimated to affect 1 in 68 children in the United States. Autism is a neurodevelopmental disorder that can impair a child’s ability to communicate and interact with others while also including restricted repetitive behaviors and interests. Although the molecular origins of these impairments are not clearly understood, studies have shown that these defects may originate from abnormal brain development during developmental stages. Topoisomerase 3B (Top3B) is an enzyme that alters the topologic states of RNA during transcription to regulate gene expression within neurons. Previous studies clearly indicate that Top3B is associated with other proteins linked to mental retardation, such as the Fragile-X mental retardation protein (FMRP). Preliminary studies with Top3B have shown that defects in Top3B could potentially result in cognitive deficits characteristic of ASD. My objective is to use CRISPR/Cas-9 as a form of molecular scissors to edit the genome of zebrafish, which would have a defective Top3B gene. This would allow me to study the effect of the defective gene on the organism at various phases of brain development. Creating a stable genetic model organism could uncover the molecular and genetic processes that govern ASD while elucidating the developmental biology. To engineer this model organism we will be using genome-editing technology. By injecting guide RNA specific to Top3b gene and a Cas9 enzyme that acts as molecular scissors, we can guide the enzyme to Top3b in single cell embryos and cut it to create a defective mutation in Top3b. Every cell replicated after this point will also carry the mutation and this will create a stable genetic line that can be inherited to the next generation. After 3-4 days post-injection we will harvest the genomic DNA from these zebrafish to ensure that our mutation of interest is present via amplification of the genetic region and sequencing. When confirming the mutation is present we will then inject multiple one-cell stage embryos to induce the mutation and study the full development of the organism as well as the next generations of zebrafish. Studies will include both developmental and behavioral observations to understand the social and physiological impact of Top3B on cognitive deficits. I am expecting the mutated model to have impaired behavior and potential phenotypic defects in neural development. This would point to mutations in Top3B as a major component of ASD type characteristics. Elucidating the genetic and molecular mechanisms for cognitive deficits associated with autism could potentially lead to targeted preventative measures while also uncovering the importance of the gene in each developmental phase.

Presentation Type and Release Option

Presentation (Open Access)

Start Date

4-16-2016 10:45 AM

End Date

4-16-2016 12:00 PM

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Apr 16th, 10:45 AM Apr 16th, 12:00 PM

Using CRISPR Based Genome Editing to Create an Autistic Zebrafish Model

Nessmith-Lane Atrium

In 2015, Autism Spectrum Disorder (ASD) was estimated to affect 1 in 68 children in the United States. Autism is a neurodevelopmental disorder that can impair a child’s ability to communicate and interact with others while also including restricted repetitive behaviors and interests. Although the molecular origins of these impairments are not clearly understood, studies have shown that these defects may originate from abnormal brain development during developmental stages. Topoisomerase 3B (Top3B) is an enzyme that alters the topologic states of RNA during transcription to regulate gene expression within neurons. Previous studies clearly indicate that Top3B is associated with other proteins linked to mental retardation, such as the Fragile-X mental retardation protein (FMRP). Preliminary studies with Top3B have shown that defects in Top3B could potentially result in cognitive deficits characteristic of ASD. My objective is to use CRISPR/Cas-9 as a form of molecular scissors to edit the genome of zebrafish, which would have a defective Top3B gene. This would allow me to study the effect of the defective gene on the organism at various phases of brain development. Creating a stable genetic model organism could uncover the molecular and genetic processes that govern ASD while elucidating the developmental biology. To engineer this model organism we will be using genome-editing technology. By injecting guide RNA specific to Top3b gene and a Cas9 enzyme that acts as molecular scissors, we can guide the enzyme to Top3b in single cell embryos and cut it to create a defective mutation in Top3b. Every cell replicated after this point will also carry the mutation and this will create a stable genetic line that can be inherited to the next generation. After 3-4 days post-injection we will harvest the genomic DNA from these zebrafish to ensure that our mutation of interest is present via amplification of the genetic region and sequencing. When confirming the mutation is present we will then inject multiple one-cell stage embryos to induce the mutation and study the full development of the organism as well as the next generations of zebrafish. Studies will include both developmental and behavioral observations to understand the social and physiological impact of Top3B on cognitive deficits. I am expecting the mutated model to have impaired behavior and potential phenotypic defects in neural development. This would point to mutations in Top3B as a major component of ASD type characteristics. Elucidating the genetic and molecular mechanisms for cognitive deficits associated with autism could potentially lead to targeted preventative measures while also uncovering the importance of the gene in each developmental phase.