Efficient generation of zebrafish knock-ins using CRISPR/Cas9
The use of CRISPR-Cas9-based genome editing to create loss-of-function alleles is nowadays common practice in zebrafish and most other model organisms. The generation of knock-in zebrafish is more challenging, and still only few studies employ this strategy to model the functional effects of human disease-associated genetic variants in zebrafish. The ability to precisely manipulate the zebrafish genome is also allows for more refined studies of fundamental cellular and developmental process in zebrafish. The introduction of specific mutations could be used to disrupt catalytic protein domains, protein-protein interactions or DNA-binding by transcription factors.
Erik de Vrieze and his colleagues from the departments of Otorhinolaryngology and Human Genetics published an improved protocol for the efficient generation of knock-in zebrafish models using CRISPR-Cas9 ribonucleoprotein complexes in the international journal of molecular sciences (IJMS). By injecting Cas9-guide RNA ribonucleoprotein complexes and a small single-stranded DNA template with the variant of interest directly in to the first cell of a fertilized zebrafish zygote, they obtain one of the highest knock-in efficiencies observed in the field. A detailed technical protocol is included in the supplementary material of the manuscript (link).
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Erik de Vrieze, Suzanne E. de Bruijn, Janine Reurink, Sanne Broekman, Vince van de Riet,
Marco Aben, Hannie Kremer and Erwin van Wijk. Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes. Int. J. Mol. Sci. 2021, 22(17):9429