Ac/Ds transposition for CRISPR/dCas9-SID4x epigenome modulation in zebrafish

Area of Science:

• Developmental Biology
• Genetics
• Epigenetics

Background:

• Zebrafish are a valuable model organism for studying (epi)genomic element function due to their genetic tractability and advancements in genome editing.
• Characterizing cis-regulated elements, such as enhancers, is crucial for understanding gene regulation.

Purpose of the Study:

• To repurpose the Ac/Ds maize transposition system for efficient characterization of zebrafish cis-regulated elements (enhancers) in F0 embryos.
• To develop a method for stable expression of guide RNAs for CRISPR/dCas9-interference (CRISPRi) to perturb enhancers without genetic disruption.
• To investigate antisense transcription at neural crest gene loci.

Main Methods:

• Utilized the Ac/Ds maize transposition system for enhancer characterization in zebrafish F0-microinjected embryos.
• Employed CRISPR/dCas9-interference (CRISPRi) for targeted perturbation of enhancer activity.
• Investigated antisense transcription at specific neural crest gene loci.

Main Results:

• Successfully repurposed the Ac/Ds transposition system for efficient characterization of zebrafish enhancers.
• Established a stable CRISPRi system for transient epigenome modulation of enhancers.
• Probed antisense transcription, providing insights into regulatory mechanisms.

Conclusions:

• The Ac/Ds transposition system offers a novel and efficient tool for transient epigenome modulation in zebrafish.
• This approach facilitates the study of enhancer function and epigenetic regulation in a live organism model.
• The developed CRISPRi system provides a non-disruptive method for functional genomics studies in zebrafish.