Identifying the Role of eve1 and vent in Zebrafish Embryogenesis with CRISPR-Cas9

Alice Dong and David Kimelman

Department of Biochemistry, University of Washington, Seattle, WA

Abstract CRISPR-Cas9 System DNA Sequencing

Gastrulation is a stage in embryogenesis in which most progenitor cells commit to a GeneWiz Next Gen Sequencing Chromatography
MOs inhibit RNA
muscle or neural fate. However, a population of bipotential progenitor cells does not
differentiate during gastrulation and allows the embryos to dynamically allocate
transcripts and can
neural and muscle tissue in the correct proportion as the body axis is formed. The cause off-target Frame Shift
regulation of this process is crucial for proper neuromuscular development. The effects
maintenance of these bipotential progenitor cells requires the repression of tbx16, a CRISPR-Cas9
transcription factor that progenitors express when they commit to a muscle fate. Here, generates efficiently
we examine two genes of interest termed eve1 and vent, and investigate their roles in and highly specific
the regulation of tbx16. While previous studies relied on Morpholino anti-sense gene knockouts. Gene sequence for identified vent fish 8 ins-1 F0 founder.
oligonucleotides (MOs), the credibility of MOs has recently become seriously
questioned because they can cause off-target effects, generating misleading effects
that don't reflect genetic mutants. Revolutionary new gene-editing technology known
CAACTCAGAGGAAG Normal sequence strand
as CRISPR, is hailed as the new standard for gene analysis because it allows the
Generating Heterozygous Mutants with CRIPSR-Cas9
CAACCTCAGAGGAA 1 bp insertion frame shift
production of true genetic mutants in specific genes. By analyzing how
embryogenesis in normal zebrafish embryos differs from that of eve1 or vent mutant
embryos, we can identify the role that these genes play during development.
Ultimately, our goal is to gain a better understanding of how serious birth defects and A Introduce mutations at gene of interest in F0 population
genetic disorders arise from complications in development.
F0 Founders

Currently, two eve1 and two vent F0 founder fish have been identified.

Wild type protein Homeobox

Introduction Inject Mutate Raise F0
eve1 fish 6 del-7 Truncated

vent & eve1 B ID and characterize founders eve1 fish 5 del-13 Truncated
Represses
tbx16 Wild type protein Homeobox
Progenitor Muscle Activates
cells Cell vent fish 4 del-4 Truncated
DNA Sequencing Raise F1
HRM vent fish 8 ins-1 Truncated

C ID F1 heterozygous mutants
Question: What role do the transcription factors Eve1 and Vent
play in regulating the genes and cellular mechanisms that are Trajectory
necessary for proper neuromuscular development?
In-situ Hybridizations
eve1 mutant embryos
Hypothesis: eve1 and vent act together to repress tbx16 and Fin Clip HRM
vent mutants embryos
prevent a population of progenitor cells from committing to a eve1 & vent double mutant embryos
muscle cell fate during embryogenesis
tbx16 staining
High Resolution Melt Analysis

Aims
Typical Wild
Type Curve Unstained Stained Stained eve1 Stained
1) Using CRISPR, generate two lines of zebrafish with an eve1 & vent single eve1/vent
Tail Bud Wild Type
gene knockout (eve1 mutants) and two lines of zebrafish with a mutants double mutant
50um

vent gene knockout (vent mutants). Predicted results: in-situ hybridizations show that eve1 and
vent act redundantly repress tbx16.
2) Test hypothesis by examining how the absence of eve1 and vent Typical Mutant
affects the expression of tbx16 in zebrafish embryos during Curve
embryogenesis.
Acknowledgement: Sponsored by the Mary Gates Scholarship Fund