Highly efficient generation of knock-in transgenic medaka by CRISPR/Cas9-mediated genome engineering

Abstract

BackgroundMedaka (Oryzias latipes) is a popular animal model used in vertebrate genetic analysis. Recently, an efficient (~ 30%) knock-in system via non-homologous end joining (NHEJ) was established in zebrafish using the CRISPR/Cas9 system. If the same technique were applicable in medaka, it would greatly expand the usefulness of this model organism. The question of the applicability of CRISPR/Cas9 in medaka, however, has yet to be addressed.ResultsWe report the highly efficient generation of knock-in transgenic medaka via non-homologous end joining (NHEJ). Donor plasmid containing a heat-shock promoter and a reporter gene was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, an sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. Broad transgene expression in the expression domain of a target gene was observed in approximately 25% of injected embryos. By raising these animals, we established stable knock-in transgenic fish with several different constructs for five genetic loci, obtaining transgenic founders at efficiencies of > 50% for all five loci. Further, we show that the method is useful for obtaining mutant alleles. In the experiments where transgene integrations were targeted between the transcription start site and the initiation methionine, the resultant transgenic fish became mutant alleles.ConclusionWith its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in via NHEJ will become a standard method for the generation of transgenic and mutant medaka.Electronic supplementary materialThe online version of this article (10.1186/s40851-017-0086-3) contains supplementary material, which is available to authorized users.

Diversity in reproductive seasonality in the three-spined stickleback,Gasterosteus aculeatus

“…Using genetically engineered fish, we will be able to investigate the pleiotropic effects of candidate genes. Finally, gene replacement has been successfully conducted in several fish species (Kimura et al, 2014;Watakabe et al, 2018), which may be possible in sticklebacks in the near future.…”

The untapped potential of medaka and its wild relatives

“…Single cell transcriptome sequencing can, for example, be used to study the evolution of gene regulatory networks underlying sex determination. All of these technologies can help to identify candidate loci that may contribute to evolutionary processes of interest, while the phenotypic effects of natural genetic variation at these loci can now be assessed using gene-editing tools (Gutierrez-Triana et al, 2018; Letelier et al, 2018; Murakami et al, 2017; Watakabe et al, 2018). Together these technological advances have set the stage for scientists to begin converting the described natural diversity of ricefishes into a better understanding of the molecular basis of evolutionary processes.…”

“…More recently, the gene-editing tool CRISPR/Cas9 was also established in medaka and employed in multiple studies (e.g. Gutierrez-Triana et al, 2018; Letelier et al, 2018; Murakami et al, 2017; Watakabe et al, 2018). In many fields, including cancer research, medaka serves as complementary model to the well-established zebrafish (Schartl and Walter, 2016; Takeda and Shimada, 2010).…”

CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome

“…CRISPR/Cas9 has been explored as a knock-in system in model fishes targeting reporter/driver genes in medaka 30,31 and zebrafish 32,33 . But limited studies for CRISPR/Cas9 knock-in were found on farmed fish 7,34 , and these studies primarily focused on non-performance genes.…”