A recombinase-activated ribozyme to knock down endogenous gene expression in zebrafish

Area of Science:

• Molecular Biology
• Gene Regulation
• Zebrafish Models

Background:

• Precise gene expression control is vital for understanding biological processes.
• Current methods using recombinases and endonucleases have variable efficiency.
• Genomic context significantly impacts the effectiveness of gene-modulating tools.

Purpose of the Study:

• To develop a novel self-cleaving ribozyme-based tool for controlling endogenous mRNA levels in zebrafish.
• To evaluate the efficiency and specificity of the T3H48 ribozyme for gene knockdown.
• To create an activatable version, RiboFlip, for conditional gene expression control.

Main Methods:

• Insertion of the T3H48 self-cleaving ribozyme into introns of target genes in zebrafish.
• Utilizing CRISPR/Cas9 and base-editing strategies for gene modification and validation.
• Development and testing of a Flippase- and Cre-activatable ribozyme (RiboFlip) with a reporter system.

Main Results:

• The T3H48 ribozyme achieved up to 20-fold reduction in target gene expression via pre-mRNA cleavage.
• RiboFlip activation successfully induced mRNA knockdown and recapitulated a pigmentation phenotype in the albino zebrafish model.
• The RiboFlip system demonstrated reversibility and allowed for cell labeling independent of target gene expression.

Conclusions:

• The RiboFlip cassette is a versatile tool for controlling endogenous gene expression in vertebrates.
• This ribozyme-based approach provides a flexible and specific alternative to existing conditional knockdown strategies.
• The study highlights the potential of ribozymes for advanced gene regulation in developmental biology and disease modeling.