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Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs.
Linlin Yin1, Lisette A Maddison1, Mingyu Li1
1Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Genetics
|April 10, 2015
Summary
This study introduces a rapid CRISPR/Cas9 system for one-generation conditional gene editing in zebrafish. This method enables multiplex gene inactivation and temporal/tissue-specific control, advancing functional genomics research.
Area of Science:
- Genetics
- Molecular Biology
- Developmental Biology
Background:
- Conditional mutagenesis is crucial for understanding gene function.
- Generating conditional alleles is challenging and inefficient, especially for multiplex studies.
- Current methods often require extensive breeding and are limited in model organisms like zebrafish.
Purpose of the Study:
- To develop a one-generation multiplex conditional mutagenesis system in zebrafish.
- To enable temporal and tissue-specific gene manipulation using CRISPR/Cas9.
- To demonstrate the efficiency and versatility of this new system for gene function studies.
Main Methods:
- Transgenic expression of Cas9 and multiple single-guide RNAs (sgRNAs) in zebrafish.
- Utilized five distinct zebrafish U6 promoters for sgRNA expression.
- Implemented heat-shock-inducible and liver-specific Cas9 expression for temporal and tissue control.
- Delivered sgRNAs targeting specific genes (tyrosinase, insulin receptor a/b, ascl1a).
Main Results:
- Achieved efficient multiplex biallelic inactivation of tyrosinase and insulin receptor genes.
- Observed defects in pigmentation and glucose homeostasis.
- Demonstrated temporal control of tyrosinase mutagenesis via heat-shock-inducible Cas9.
- Showcased tissue-specific disruption of insulin receptors in the liver, leading to metabolic dysregulation.
- Impaired photoreceptor regeneration in the eye after targeting ascl1a.
Conclusions:
- CRISPR/Cas9-based conditional mutagenesis is feasible, rapid, and straightforward in zebrafish.
- This system significantly enhances the study of gene function through efficient multiplex and conditional editing.
- Offers a powerful new tool for genetic research in zebrafish, overcoming previous limitations.
