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Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish
Published on: July 22, 2025
692
Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs.
Brian H Kim1, GuangJun Zhang2,3,4,5
1Department of Comparative Pathobiology.
G3 (Bethesda, Md.)
|January 10, 2020
Summary
Multiplex CRISPR-Cas9 injection enables large DNA deletions and exon removal in zebrafish, facilitating the creation of effective loss-of-function mutants for complex gene studies.
Area of Science:
- Genetics
- Molecular Biology
- Developmental Biology
Background:
- CRISPR-Cas9 is a powerful genetic tool for gene editing.
- Single guide RNA (gRNA) typically induces small indel mutations.
- Alternative splicing from small mutations can complicate loss-of-function studies.
Purpose of the Study:
- To develop a method for generating null or null-like loss-of-function mutants in zebrafish.
- To overcome challenges posed by alternative splicing in CRISPR-Cas9 gene editing.
Main Methods:
- Simultaneous injection of multiple gRNAs into single-cell stage zebrafish embryos.
- Utilizing multiplex gRNA strategy for large-scale genomic deletions.
Main Results:
- Demonstrated deletion of large DNA segments (up to 78kb) in the smarca2 gene locus.
- Successfully induced multiple exon deletions in rnf185 and rnf215 genes in F1 zebrafish.
- Achieved efficient gene mutation using multiplex gRNA injection.
Conclusions:
- Multiplex gRNA injection is effective for creating substantial genomic alterations in zebrafish.
- This approach is valuable for generating robust loss-of-function mutants, especially for genes with unclear phenotypes.
- The strategy is applicable to other vertebrate organisms for knock-out studies.
