Zebrafish knock-ins swim into the mainstream

Abstract

The zebrafish is an increasingly popular model organism for human genetic disease research. CRISPR/Cas9-based approaches are currently used for multiple gene-editing purposes in zebrafish, but few studies have developed reliable ways to introduce precise mutations. Point mutation knock-in using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) is currently the most promising technology for this purpose. Despite some progress in applying this technique to zebrafish, there is still a great need for improvements in terms of its efficiency, optimal design of sgRNA and ssODNs and broader applicability. The papers discussed in this Editorial provide excellent case studies on identifying problems inherent in the mutation knock-in technique, quantifying these issues and proposing strategies to overcome them. These reports also illustrate how the procedures for introducing specific mutations can be straightforward, such that ssODNs with only the target mutation are sufficient for generating the intended knock-in animals. Two of the studies also develop interesting point mutant knock-in models for cardiac diseases, validating the translational relevance of generating knock-in mutations and opening the door to many possibilities for their further study.

Zebrafish as an Emerging Model for Osteoporosis: A Primary Testing Platform for Screening New Osteo-Active Compounds

“…Moreover, the CRISPR/Cas9 protocol is so efficient that the F0 injected fish (crispants) can be used to study loss of gene function in these crispants, despite them carrying mosaic mutations (i.e., not every cell carries a mutation and more than one mutation may be present) (23, 60) (Figure 1B). Single base gene editing (knock ins) using modified Cas9 enzymes or supplying a DNA template for the endogenous homologous recombination machinery initiated by a double stranded break allows to introduce specific genetic changes to model specific human disease mutations in zebrafish orthologs (62, 63).…”

The zebrafish subcortical social brain as a model for studying social behavior disorders

“…CRISPR is a popular technology for genome editing in zebrafish due to its simplicity and speed (Hwang et al, 2013; Prykhozhij et al, 2017; Prykhozhij and Berman, 2018). Researchers have attempted to improve throughput by developing more scalable methods.…”

Inducible modulation of miR-204 levels in a zebrafish melanoma model

“…They can be adapted to virtually any experimental need, by choosing the most suitable promoter (for total body or tissue-specific expression); gene of interest (non-coding, but also coding. Of note, besides sponge-204 or pre-miR-204, the vectors here described also express the coding eGFP mRNA, for a total transcript length of ∼1 kb); mechanism and degree of gene modulation [overexpression ( Jung et al, 2019 ); downregulation using RNA interference ( Giacomotto et al, 2015 ); knock in ( Prykhozhij and Berman, 2018 ) or knock out ( Kaufman et al, 2016 ; Ablain et al, 2018 ; Jung et al, 2019 ) using CRISPR/Cas9 technology].…”