Journal Article

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

van de Riet, Vince, van Wijk, Erwin, Reurink, Janine, Aben, Marco, Kremer, Hannie, de Vrieze, Erik, de Bruijn, Suzanne E., Broekman, Sanne

2021

Overview

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.

• Publisher: MDPI AG,MDPI
• Subject: Animals / CRISPR / CRISPR-Cas Systems / Disease Models, Animal / DNA repair / Efficiency / Embryos / Gene Editing / Gene Knock-In Techniques - methods / Genetic Diseases, Inborn - genetics / Genetic engineering / Genetic Engineering - methods / Genome editing / Genomes / Mutagenesis / Mutation / Proteins / Ribonucleoproteins - genetics / Ribonucleoproteins - metabolism / Zebrafish / Zebrafish Proteins - antagonists & inhibitors / Zebrafish Proteins - genetics / Zebrafish Proteins - metabolism