Efficient generation of knock‐in zebrafish models for inherited disorders using crispr‐cas9 ribonucleoprotein complexes

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.