A Rapid Method for Directed Gene Knockout for Screening in G0 Zebrafish

Publication date: 2 July 2018
Source:Developmental Cell, Volume 46, Issue 1
Author(s): Roland S. Wu, Ian I. Lam, Hilary Clay, Daniel N. Duong, Rahul C. Deo, Shaun R. Coughlin
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.

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Teaser

Wu et al. describe a pipeline for CRISPR/Cas9 genetic screening with optimized, redundant gene targeting to produce penetrant gene disruption in zebrafish. The authors evaluate this system on several genes and apply this strategy to 50 empirically identified zebrafish cardiomyocyte transcription factors, uncovering a role for zbtb16a in cardiac development.


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