PUBLICATION
A Rapid Method for Directed Gene Knockout for Screening in G0 Zebrafish
- Authors
- Wu, R.S., Lam, I.I., Clay, H., Duong, D.N., Deo, R.C., Coughlin, S.R.
- ID
- ZDB-PUB-180706-3
- Date
- 2018
- Source
- Developmental Cell 46: 112-125.e4 (Journal)
- Registered Authors
- Clay, Hilary, Deo, Rahul C., Shaun Coughlin
- Keywords
- CRISPR, Cas9, G0, gene editing, mutagenesis, reverse genetic screen, zebrafish
- Datasets
- GEO:GSE115263, GEO:GSE115233
- MeSH Terms
-
- Animals
- Base Sequence
- CRISPR-Cas Systems/genetics
- Clustered Regularly Interspaced Short Palindromic Repeats/genetics
- GTP-Binding Protein alpha Subunits, G12-G13/genetics
- Gene Knockout Techniques/methods*
- Genetic Engineering/methods
- Heart/embryology*
- Morpholinos/genetics
- Myocytes, Cardiac/cytology
- Promyelocytic Leukemia Zinc Finger Protein/genetics*
- Transcription, Genetic/genetics
- Zebrafish/embryology
- Zebrafish/genetics*
- Zebrafish Proteins/genetics*
- PubMed
- 29974860 Full text @ Dev. Cell
Citation
Wu, R.S., Lam, I.I., Clay, H., Duong, D.N., Deo, R.C., Coughlin, S.R. (2018) A Rapid Method for Directed Gene Knockout for Screening in G0 Zebrafish. Developmental Cell. 46:112-125.e4.
Abstract
Zebrafish is a powerful model for forward genetics. Reverse genetic approaches are limited by the time required to generate stable mutant lines. We describe a system for gene knockout that consistently produces null phenotypes in G0 zebrafish. Yolk injection of sets of four CRISPR/Cas9 ribonucleoprotein complexes redundantly targeting a single gene recapitulated germline-transmitted knockout phenotypes in >90% of G0 embryos for each of 8 test genes. Early embryonic (6 hpf) and stable adult phenotypes were produced. Simultaneous multi-gene knockout was feasible but associated with toxicity in some cases. To facilitate use, we generated a lookup table of four-guide sets for 21,386 zebrafish genes and validated several. Using this resource, we targeted 50 cardiomyocyte transcriptional regulators and uncovered a role of zbtb16a in cardiac development. This system provides a platform for rapid screening of genes of interest in development, physiology, and disease models in zebrafish.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping