An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes)

Murakami, Yu; Ansai, Satoshi; Yonemura, Akari; Kinoshita, Masato

doi:10.1186/s40851-017-0071-x

Cited by 34 publications

(46 citation statements)

References 37 publications

(61 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The NHEJ-mediated knock-in method described here has one clear advantage over the homology-based recombination technique that was recently described by [ 5 ]. The current method does not require any DNA construction experiments.…”

Section: Discussionmentioning

confidence: 99%

“…As shown in Additional file 4 : Figure S2, indels were frequently introduced in the joint region, similar to the previous study [ 18 ]. For experiments in which precise knock-in is critical, methods that rely on homology-dependent repair systems (i.e., [ 5 ]) need to be employed.…”

Section: Discussionmentioning

confidence: 99%

“…Traditional methods for the generation of transgenic medaka, however, require promoter/enhancer hunting or bacterial artificial chromosome (BAC) modification, both of which involve time-consuming steps. Recently, the targeted knock-in of a reporter construct via a homology-dependent DNA repair was shown to work well in medaka using the CRISPR/Cas9 system [ 5 ]. Although the technique is ideal for precise knock-in, it also requires time-consuming molecular cloning steps for the construction of a donor plasmid.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

et al. 2018

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2018

View full text Add to dashboard Cite

show abstract

“…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).…”

Section: Introductionmentioning

confidence: 99%

The untapped potential of medaka and its wild relatives

Hilgers

Schwarzer

2019

eLife

View full text Add to dashboard Cite

The medaka is a fish that has served as a model organism for over a century, yet there is still much to learn about its life in the wild. Here we summarize the current knowledge, highlight recent progress and outline remaining gaps in our understanding of the natural history of medaka. It has also become clear over time that rather than being a single species, medaka comprises an entire species complex, so disentangling the species boundaries is an important goal for future research. Moreover, medaka and other ricefishes exhibit striking functional diversity, little of which has been investigated to date. As such, there are opportunities to use the resources developed for medaka to study other ricefishes, and to learn more about medaka itself in an evolutionary context.

show abstract

“…With the exception of very low efficiency KI of a gene encoding red fluorescent protein in rohu carp 34 , such advanced and fine-tuned genome editing has not been developed for farmed fish, and it may be useful to learn from protocols already established in model fish species. While only a few studies have reported HDR in medaka 35,36 , several KI-strategies have been reported in zebrafish using either donor plasmids [37][38][39] or single-stranded oligodeoxynucleotides (ssODNs) [40][41][42][43][44][45] , or both 46 . Knowledge from other fish studies are somewhat inconclusive when it comes to deciding the strategy for applying HDR in salmon, as there is a lack of consensus regarding the impact of different repair templates, homology arm length and strand complementarity in the above-mentioned studies.…”

mentioning

confidence: 99%

Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9-mediated HDR in Atlantic salmon

Straume

Kjærner-Semb

Skaftnesmo

et al. 2020

Sci Rep

View full text Add to dashboard Cite

Precise gene editing such as CRISPR/Cas9-mediated homology directed repair (HDR) can increase our understanding of gene function and improve traits of importance for aquaculture. This fine-tuned technology has not been developed for farmed fish including Atlantic salmon. We performed knock-in (KI) of a FLAG element in the slc45a2 gene in salmon using sense (S), anti-sense (AS) and doublestranded (ds) oligodeoxynucleotide (ODN) templates with short (24/48/84 bp) homology arms. We show in vivo ODN integration in almost all the gene edited animals, and demonstrate perfect HDR rates up to 27% in individual F0 embryos, much higher than reported previously in any fish. HDR efficiency was dependent on template concentration, but not homology arm length. Analysis of imperfect HDR variants suggest that repair occurs by synthesis-dependent strand annealing (SDSA), as we show for the first time in any species that indel location is dependent on template polarity. Correct ODN polarity can be used to avoid 5′-indels interrupting the reading frame of an inserted sequence and be of importance for HDR template design in general. Aquaculture continues to grow faster than any other major food production sector and is quickly becoming the main source of seafood in human diets. In this context, Norway is the largest producer of farmed Atlantic salmon (Salmo salar) worldwide. In later years, the production of salmon in Norway has ceased to grow due to sustainability challenges linked to open sea-cage rearing. Genetic introgression of farmed salmon into wild stocks and the marine parasite, salmon louse, are recognized as the two major concerns 1. The high prevalence of salmon lice in most Norwegian fjords, due to open sea-cage farming, cause high lethality in wild salmonids and is hindering expansion of sea-cage farming. The consequences of genetic introgression caused by escapees remain uncertain, but existing knowledge indicates that it may lead to changes in life-history traits, with potential ecological impacts 2-5. Sequencing of the salmon genome 6 has permitted more detailed studies on the link between genes and key traits, and we and others have shown that single nucleotide polymorphisms (SNPs) to a certain degree can explain the time of maturity 1 and disease resistance 7,8. In this context, New Breeding Technologies (NBTs) by gene editing may offer a solution to some of the problems in salmon farming, with a possible production of salmon displaying traits such as disease resistance and sterility 9-12. We have previously demonstrated the feasibility of double allelic KO in F0 salmon using CRISPR/Cas9, by targeting genes essential for pigmentation 9 , elongation of polyunsaturated fatty acids 13 and reproduction 10. At the same time, CRISPR/Cas9 KO-mutations targeting various phenotypes have been shown by others in several farmed fish species such as tilapia 14-21 , sea bream 22 , sterlet 23 , channel catfish 24,25 , southern catfish 26 , common carp 27 , sturgeon 28 and rainbow trout 29. CRISPR/Cas9 KOs are produced by a Cas9-...

show abstract

An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes)

Cited by 34 publications

References 37 publications

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

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

The untapped potential of medaka and its wild relatives

Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9-mediated HDR in Atlantic salmon

Contact Info

Product

Resources

About