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.

A robust pipeline for efficient knock-in of point mutations and epitope tags in zebrafish using fluorescent PCR based screening

“…Our methods present several advantages over the commonly used sequencing-based screening approaches. First, our methods are based on fluorescent PCR [ 6 ] and CRISPR-STAT [ 34 ], both of which are becoming popular in the zebrafish community for accurate genotyping of fish with indels and evaluation of sgRNAs, respectively [ 21 , 52 – 60 ]. Thus, it would be easy to implement them for knock-in screening as described here.…”

“…While indels at the 5’ end of ssODN integration site would lead to a change in the size of the digested fragment, indels at the 3’ end are missed as it gets cleaved off after the digest. However, unlike allele-specific PCR [ 24 ] or NGS based screening methods [ 11 , 17 , 21 , 24 , 60 ] that require additional validation steps, our primers are designed to not overlap with the ssODN and therefore, we could simply sequence the PCR product to confirm precise or imprecise integration events without additional amplification steps. Thus, although there is a caveat to our screening approach for point mutations, it can be overcome by screening additional prioritized founders and sequence validation.…”

“…Screening for precise knock-in is especially difficult when using ssODNs due to the lack of visual tools, like fluorescent reporters, which can be used for knock-in with larger repair templates [ 26 , 30 , 31 ]. Therefore, expensive and labor-intensive approaches, such as cloning and sequencing of a large number of clones or next-generation sequencing (NGS) of pooled embryos are used to determine the success of knock-in using ssODNs [ 4 , 11 , 17 , 21 , 23 , 24 ]. NGS requires access to special equipment and bioinformatic expertise for processing the large amounts of sequencing data, thus posing a challenge for many laboratories.…”

Zebrafish, Medaka and Turquoise Killifish for Understanding Human Neurodegenerative/Neurodevelopmental Disorders

“…The CRISPR-Cas9 system also enables knock-in of the target gene or specific gene variants by means of homology-directed repair (HDR) or other mechanisms [45][46][47][48][49][50]. Stable introduction of exogenous DNA in the zebrafish genome was already achieved in 1988 [51].…”

Zebrafish as a Model to Study Vascular Elastic Fibers and Associated Pathologies

“…The CRISPR technique has also been developed to perform knockins and conditional knockouts in the zebrafish [15,16]. These modifications can be helpful to implement models with relevant characteristics of human diseases, such as specific variants associated with pathologies [17], restriction of genetic modifications to a precise time period and/or location [18], and also to avoid the compensations that are often associated with knockout experiments [19].…”