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.
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
Microarrays: GEO:GSE115233, GEO:GSE115263
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
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.