PUBLICATION

Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes

Authors
de Vrieze, E., de Bruijn, S.E., Reurink, J., Broekman, S., van de Riet, V., Aben, M., Kremer, H., van Wijk, E.
ID
ZDB-PUB-210911-12
Date
2021
Source
International Journal of Molecular Sciences   22(17): (Journal)
Registered Authors
Aben, Marco, de Bruijn, Ewart, de Vrieze, Erik, Kremer, Hannie, van Wijk, Erwin
Keywords
CRISPR-Cas9, disease models, homology-directed repair, knock-in, zebrafish
MeSH Terms
  • Animals
  • CRISPR-Cas Systems*
  • Disease Models, Animal*
  • Gene Editing*
  • Gene Knock-In Techniques/methods*
  • Genetic Diseases, Inborn/genetics*
  • Genetic Engineering/methods*
  • Mutagenesis
  • Ribonucleoproteins/genetics
  • Ribonucleoproteins/metabolism
  • Zebrafish
  • Zebrafish Proteins/antagonists & inhibitors
  • Zebrafish Proteins/genetics*
  • Zebrafish Proteins/metabolism
PubMed
34502338 Full text @ Int. J. Mol. Sci.
Abstract
CRISPR-Cas9-based genome-editing is a highly efficient and cost-effective method to generate zebrafish loss-of-function alleles. However, introducing patient-specific variants into the zebrafish genome with CRISPR-Cas9 remains challenging. Targeting options can be limited by the predetermined genetic context, and the efficiency of the homology-directed DNA repair pathway is relatively low. Here, we illustrate our efficient approach to develop knock-in zebrafish models using two previously variants associated with hereditary sensory deficits. We employ sgRNA-Cas9 ribonucleoprotein (RNP) complexes that are micro-injected into the first cell of fertilized zebrafish eggs together with an asymmetric, single-stranded DNA template containing the variant of interest. The introduction of knock-in events was confirmed by massive parallel sequencing of genomic DNA extracted from a pool of injected embryos. Simultaneous morpholino-induced blocking of a key component of the non-homologous end joining DNA repair pathway, Ku70, improved the knock-in efficiency for one of the targets. Our use of RNP complexes provides an improved knock-in efficiency as compared to previously published studies. Correct knock-in events were identified in 3-8% of alleles, and 30-45% of injected animals had the target variant in their germline. The detailed technical and procedural insights described here provide a valuable framework for the efficient development of knock-in zebrafish models.
Genes / Markers
Figures
Show all Figures
Expression
Phenotype
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping
Errata and Notes