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.
Highlights
The use of CRISPR-Cas9-based genome editing to create loss-of-function alleles is nowadays common practice in most model organisms
One of the easiest animal models to manipulate with CRISPR-Cas9, precise editing using a template for homology directed repair (HDR) was first reported by Irion and colleagues in 2014 [3]
We previously described the generation of a humanized zebrafish knock-in model for the pathogenic deep-intronic c.7595-2144A>G variant in human USH2A, resulting in the inclusion of a pseudo-exon (PE40)
Summary
The use of CRISPR-Cas9-based genome editing to create loss-of-function alleles is nowadays common practice in most model organisms. While the first reports employing the CRISPR-Cas system to knock-in specific genetic variants by means of homology directed repair (HDR) date back to 2013 [1,2], the overall efficiency of this application remains much lower as compared to the generation of knock-out models. One of the easiest animal models to manipulate with CRISPR-Cas, precise editing using a template for HDR was first reported by Irion and colleagues in 2014 [3]. They used a circular template vector containing 875 bp homology arms flanking the variant of interest, which they co-injected with Cas mRNA and in-vitro synthesized sgRNA molecules to repair the albino mutation (alb allele).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
