|ZFIN ID: ZDB-PUB-180706-3|
A Rapid Method for Directed Gene Knockout for Screening in G0 Zebrafish
Wu, R.S., Lam, I.I., Clay, H., Duong, D.N., Deo, R.C., Coughlin, S.R.
|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|
|PubMed:||29974860 Full text @ Dev. Cell|
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.
ABSTRACTZebrafish 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.