Versatile CRISPR-Cas Tools for Gene Regulation in Zebrafish via an Enhanced Q Binary System

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• CRISPR-Cas systems offer powerful gene regulation but zebrafish studies often use transient delivery, limiting transgenic applications.
• Existing transgenic CRISPR models face challenges with sustained, tissue-specific expression and potential toxicity or transgene silencing.

Purpose of the Study:

• To develop a robust transgenic CRISPR system for sustained and spatiotemporal gene regulation in zebrafish.
• To overcome limitations of transient delivery and other binary systems for precise gene manipulation in zebrafish models.

Main Methods:

• Developed CRISPR-Q, integrating the QFvpr/QUAS binary system with CRISPR-Cas technologies (CasRx, dCas9vpr).
• Utilized CRISPR-Q for transcript knockdown (CRISPR-Q_KD) and gene activation (CRISPR-Qa) in zebrafish.
• Validated tissue-specific expression in heart-specific transgenic zebrafish.

Main Results:

• CRISPR-Q enabled robust spatiotemporal expression of CRISPR effectors, achieving precise transcript knockdown.
• Successfully modeled spinal muscular atrophy and amyotrophic lateral sclerosis by knocking down specific genes (smn1, tardbp, tardbpl).
• Demonstrated gene activation by inducing endogenous lin28a and sox9b expression using CRISPR-Qa.

Conclusions:

• CRISPR-Q is a versatile and efficient transgenic platform for gene function studies and disease modeling in zebrafish.
• The system overcomes previous limitations, offering broad potential for adaptation in other model organisms.