PUBLICATION
Generation of Transgenic Fish Harboring CRISPR/Cas9-Mediated Somatic Mutations Via a tRNA-Based Multiplex sgRNA Expression
- Authors
- Shiraki, T., Kawakami, K.
- ID
- ZDB-PUB-230906-70
- Date
- 2024
- Source
- Methods in molecular biology (Clifton, N.J.) 2707: 305318305-318 (Chapter)
- Registered Authors
- Kawakami, Koichi, Shiraki, Tomoya
- Keywords
- CRISPR/Cas9, Genome editing, Somatic mutations, Transgenic, sgRNA, tRNA
- MeSH Terms
-
- Animals
- Animals, Genetically Modified/genetics
- CRISPR-Cas Systems/genetics
- Frameshift Mutation
- RNA, Guide, CRISPR-Cas Systems*
- Zebrafish*/genetics
- PubMed
- 37668921 Full text @ Meth. Mol. Biol.
Citation
Shiraki, T., Kawakami, K. (2024) Generation of Transgenic Fish Harboring CRISPR/Cas9-Mediated Somatic Mutations Via a tRNA-Based Multiplex sgRNA Expression. Methods in molecular biology (Clifton, N.J.). 2707:305318305-318.
Abstract
The controlled expression of Cas9 and/or sgRNA in transgenic zebrafish made it possible to knock out a gene in a spatially and/or temporally controlled manner. This transgenic approach can be more useful if multiple sgRNAs are efficiently expressed since we can improve the biallelic frame-shift mutation rate and circumvent the functional redundancy of genes and genetic compensation. We developed the tRNA-based system to express multiple functional sgRNAs from a single transcript in zebrafish and found that it is applicable to the transgenic expression of multiple sgRNAs. In this chapter, we describe a procedure for the generation of plasmids containing multiple sgRNAs flanked by tRNAs and a method to induce multiple CRISPR/Cas9-mediated genome modifications in transgenic zebrafish.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping