Introduction: Genome editing using CRISPR/Cas9 has advanced very rapidly in its scope,
versatility and ease of use. Zebrafish (Danio rerio) has been one of the vertebrate model species where
CRISPR/Cas9 has been applied very extensively for many different purposes and with great success.
In particular, disease modeling in zebrafish is useful for testing specific gene variants for pathogenicity
in a preclinical setting. Here we describe multiple advances in diverse species and systems that can
improve genome editing in zebrafish.
Objective: To achieve temporal and spatial precision of genome editing, many new technologies can
be applied in zebrafish such as artificial transcription factors, drug-inducible or optogenetically-driven
expression of Cas9, or chemically-inducible activation of Cas9. Moreover, chemically- or optogenetically-
inducible reconstitution of dead Cas9 (catalytically inactive, dCas9) can enable spatiotemporal
control of gene regulation. In addition to controlling where and when genome editing occurs, using
oligonucleotides allows for the introduction (knock-in) of precise modifications of the genome.
Conclusion: We review recent trends to improve the precision and efficiency of oligo-based point mutation
knock-ins and discuss how these improvements can apply to work in zebrafish. Similarly to how
chemical mutagenesis enabled the first genetic screens in zebrafish, multiplexed sgRNA libraries and
Cas9 can enable the next revolutionary transition in how genetic screens are performed in this species.
We discuss the first examples and prospects of approaches using sgRNAs as specific and effective
mutagens. Moreover, we have reviewed methods aimed at measuring the phenotypes of single cells
after their mutagenic perturbation with vectors encoding individual sgRNAs. These methods can range
from different cell-based reporters to single-cell RNA sequencing and can serve as great tools for
high-throughput genetic screens.