Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes
- de Vrieze, E., de Bruijn, S.E., Reurink, J., Broekman, S., van de Riet, V., Aben, M., Kremer, H., van Wijk, E.
- International Journal of Molecular Sciences 22(17): (Journal)
- Registered Authors
- Aben, Marco, de Bruijn, Ewart, de Vrieze, Erik, Kremer, Hannie, van Wijk, Erwin
- CRISPR-Cas9, disease models, homology-directed repair, knock-in, zebrafish
- MeSH Terms
- CRISPR-Cas Systems*
- Disease Models, Animal*
- Gene Editing*
- Gene Knock-In Techniques/methods*
- Genetic Diseases, Inborn/genetics*
- Genetic Engineering/methods*
- Zebrafish Proteins/antagonists & inhibitors
- Zebrafish Proteins/genetics*
- Zebrafish Proteins/metabolism
- 34502338 Full text @ Int. J. Mol. Sci.
de Vrieze, E., de Bruijn, S.E., Reurink, J., Broekman, S., van de Riet, V., Aben, M., Kremer, H., van Wijk, E. (2021) Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes. International Journal of Molecular Sciences. 22(17):.
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
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes