Highly efficient generation of knock-in transgenic medaka by CRISPR/Cas9-mediated genome engineering
© The Author(s). 2018
Received: 12 September 2017
Accepted: 29 December 2017
Published: 5 February 2018
Medaka (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.
We 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.
With 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.
Medaka (Oryzias latipes) is a small freshwater teleost species. Similar to zebrafish (Danio rerio), medaka is a popular animal model for vertebrate genetic analysis and offers many advantages, including the availability of highly polymorphic inbred strains that can be effectively used for genetic mapping [1–3].
Transgenic animals with reporter expression in specific tissues or cell types are valuable tools, and many transgenic strains have been generated in medaka [1, 4]. 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 . Although the technique is ideal for precise knock-in, it also requires time-consuming molecular cloning steps for the construction of a donor plasmid. In zebrafish, an efficient (~ 30%) knock-in system via non-homologous end joining (NHEJ) has been established . In this method, the co-injection of donor plasmid, short guide RNAs (sgRNAs), and Cas9 mRNA lead to the concurrent digestion of the genomic DNA and the donor plasmid, resulting in the incorporation of the donor plasmid into the genome. The technique does not require molecular cloning steps for the construction of a donor plasmid, and is now becoming standard for the generation of transgenic fish with reporter gene expression in a specific tissue. The technique has also been shown to be useful for the generation of mutant alleles . If the same technique were shown to be applicable in medaka, it would greatly expand the usefulness of this model organism. To date, however, this question has remained unresolved.
We show that reporter constructs consisting of a medaka heat shock promoter (expected to work as a minimum promoter) and reporter genes integrated into the aimed genomic loci with high frequency via CRISPR/Cas9-mediated NHEJ; more than 50% of raised animals became transgenic founders. We further show that integrations can lead to the disruption of a gene when the integration was targeted between the transcription start site and the initiation methionine. Given 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.
Fish care and strains
Medaka adults, embryos, and larvae were maintained at 25–28 °C. All procedures were performed in compliance with the guidelines approved by the animal care and use committees of the National Institutes of Natural Sciences and Nagoya University. Animals were staged by days post fertilization (dpf). The parental strain for the generation of all transgenic fish was d-rR. The genetic background was Nagoya for ml-3 (sox5), orange-red variety for lf-2 (pax7a), and d-rR for pnp4a.
Construction of donor DNA for knock-in
Tbait (GGCTGCTGTCAGGGAGCTCATGG) sequence  was used as a bait sequence in donor plasmids. Two types of donor plasmids were used in this study: Tbait-hs-loxP-RFP-loxP-GFP and Tbait-hs-GFP. The hs represents the 0.8 kb sequence from the medaka hsp70 promoter (the hsp70.1 gene; ). The sequence was amplified by polymerase chain reaction (PCR) with primers: AGCTGCGTCACGTGGTCCCG (forward) and TGCTTTGTGCTGTAAAGACGC (reverse). Except for the usage of the medaka hs promoter, the constituents of Tbait-hs-loxP-RFP-loxP-GFP and Tbait-hs-GFP plasmids are essentially as described in  and .
Construction of zhspa8:Cre and generation of Tg[zhspa8:Cre-mCherry-NLS] transgenic fish
Zebrafish hspa8 promoter, approximately 2.6 kb in length [9, 10], was used to express Cre-mCherry-NLS  ubiquitously in early embryos. The zhsp8 promoter, Cre-mCherry-NLS, and bovine growth hormone (BGH) polyA sequences were placed in this order in the Tol2-based vector, pT2KXIGΔin . Microinjection of Tol2-based plasmid DNA into medaka embryos was performed as was done in zebrafish .
Preparation of sgRNAs
For the vacht and nr5a1 genes, we tested two sgRNAs. The sgRNA that yielded best results in F0 expression assay was chosen to generate stable transgenic fish (Table 1). For the sox5, pax7a, and pnp4a genes, we tested just one sgRNA for each gene (Table 1).
Microinjection for knock-in
Results of microinjections and screening of transgenic founders
survived at 7dpf
survived to adult
5 / 10 (50%)
1 / 1 (100%)
7/ 10 (70%)
6 / 7 (85.7%)
3 / 6 (50%)
23 / 34 (67.6%)
For insertion mapping, fluorescent F1 animals at 5–9 dpf were collected, and genomic DNA was extracted with standard protocols. The insertion status was examined on either the 5′ side or the 3′ side of the insertion. For example, to examine the 5′ side of the insertion, a PCR reaction was performed using a 5′ primer that was specific to each gene (upstream of the expected insertion site) and a 3′ primer that was specific to the donor plasmid (sequence within the hsp70 promoter for detecting the forward insertion, and sequence within pBluescriptSK for detecting the inverse insertion). To examine whether the tandem-array insertion in the same direction occurred, a PCR reaction was performed with the two primers within the donor plasmid.
For the sox5 and pax7a transgenic fish, nucleotide sequences of PCR products were determined to examine the joint regions of the insertions.
Images were taken using an MVX10 microscope (Olympus, Tokyo, Japan), an MZ APO stereomicroscope (Leica, Wetzlar, Germany), and an LSM700 confocal laser-scanning microscope (Zeiss, Oberkochen, Germany).
Strategy for the generation of knock-in medaka and generation of Tg[vacht-hs:lRl-GFP] strains
We tested two sgRNAs (vacht-sg1 and vacht-sg2; Table 1). Injected animals were investigated for their RFP expression around the hatching stage. In the case of vacht-sg1, we found animals that showed wide-spread RFP expression in the trunk motoneurons (Fig. 1d; 1c is a control animal) in 55% (11 of 20) of the animals (Table 2). The remaining 45% of the animals either showed sparse RFP expression in the motoneurons (Fig. 1e) or no expression. In the zebrafish experiments, there was a strong correlation between the expression levels of a reporter gene in injected animals and the probability of becoming transgenic founders. Animals that had good reporter gene expression had a high probability of becoming positive founders . Thus, we raised only those animals that were considered to have “good expression” (Fig. 1d; Table 2). The raised animals were crossed to wild-type to examine if they would produce fluorescent offspring. Among 10 fish screened, five produced larvae with RFP expression in the motoneurons (Fig. 1f; Table 2). The expression patterns of RFP in these Tg [vacht-hs:lRl-GFP] transgenic fish were similar among progenies from different founders. We investigated the insertion sites of each line by PCR, and found that, in all cases, the transgene was integrated around the expected site in the genome. As was seen in zebrafish, both forward-direction integration and reverse-direction integration were observed. Moreover, in some cases multiple copies of donor plasmid were integrated (Additional file 2: Table S2).
The medaka hsp70 promoter employed in this study showed activity in female germ cells. Embryos produced from transgenic females showed ubiquitous red fluorescence due to the maternal effect (Fig. 1g). Expression levels were variable even among progenies from the same female (Fig. 1g). This maternally-derived fluorescence became negligible at around 3 dpf, and thus did not represent a major problem for observation of RFP-labeled motoneurons at later stages. The maternally derived fluorescence was also observed in other lines generated in this study.
Conversion of RFP transgenic fish to GFP transgenic fish by crossing
The reporter sequence (loxP-RFP-loxP-GFP) in the Tg[vacht-hs:lRl-GFP] fish described above was aimed such that RFP expression could be converted to GFP expression by the application of Cre. To conveniently change RFP transgenic fish to GFP transgenic fish, we generated transgenic medaka that ubiquitously express Cre-mCherry-NLS fusion protein  in early embryos. For this purpose, we used the zebrafish hspa8 promoter, which is known to drive gene expression ubiquitously in early zebrafish embryos [9, 10].
Generation of Tg[nr5a1-hs:lRl-GFP] and Tg[nr5a1-hs:GFP] strains
We then examined if RFP fish could be converted to GFP fish by crossing. A Tg[nr5a1-hs:lRl-GFP] transgenic founder was crossed to Tg[zhsp8:Cre-mCherry-NLS] fish. This resulted in the production of animals in which GFP instead of RFP was expressed in the hypothalamus (hp), interrenal gland (ir), and gonad (g) (Figs. 3e and f). The animals were raised to adulthood, and were then verified to produce GFP-expressing fish. These GFP transgenic fish did not express RFP (Figs. 3e' and f'). Thus, RFP transgenic fish were converted to GFP transgenic fish, resulting in the establishment of Tg[nr5a1-hs:GFP].
Generation of mutant alleles for pax7a and sox5 genes by NHEJ-mediated knock-in
In the knock-in experiments for the vacht and nr5a1 genes, we did not aim to disrupt gene functions. The insertion sites were set upstream of the genes, not in the exons. In zebrafish studies, the NHEJ-mediated knock-in technique has also shown to be effective for obtaining mutant alleles by inserting the transgene into exons . This led us to examine whether this is also the case in medaka.
Efficient generation of transgenic zebrafish with CRISPR/Cas9
In zebrafish, we have previously shown that knock-in transgenic fish can be efficiently generated via NHEJ by co-injection of two sgRNAs (one for the digestion of the genome and the other for the digestion of donor plasmid), donor plasmid, and Cas9 mRNA. Here, we have shown that the same method is perfectly applicable in medaka. Injected animals frequently show reporter gene expression broadly in cells where targeted genes are expressed (frequency, 30–50% for three of the five genes (vacht, sox5, and pax7a) and 10–20% for the other two genes (nr5a1 and pnp4a); in total, 27% (47 of 177); Table 2). By raising those animals to adulthood, we were able to obtain transgenic founders with very high frequency. Efficiencies exceeded 50% for all five genes (Table 2). Thus, we have succeeded in establishing a highly-efficient knock-in system in medaka fish.
The frequency obtained in this study was higher than that in zebrafish where 5–10% of injected animals usually show “good expression”, and approximately 30% of raised animals became positive founders . One potential reason that accounts for these higher frequencies is the slower development of medaka embryos compared to zebrafish embryos [20, 21]: In medaka, there may be more opportunities for the donor plasmid to integrate in the very early developmental stages.
The NHEJ-mediated knock-in method described here has one clear advantage over the homology-based recombination technique that was recently described by . The current method does not require any DNA construction experiments. By contrast, recombination techniques based on long-homology arms require DNA construction steps. One clear disadvantage of the NHEJ-mediated knock-in system is that it is not suitable for precise knock-in. As shown in Additional file 4: Figure S2, indels were frequently introduced in the joint region, similar to the previous study . For experiments in which precise knock-in is critical, methods that rely on homology-dependent repair systems (i.e., ) need to be employed.
Generation of mutant alleles with NHEJ-mediated knock-in
In this study, we demonstrated that mutant alleles can be efficiently generated by NHEJ-mediated knock-in. For all three of the genes tested, we succeeded in obtaining mutant alleles with high frequency (Table 2). The basic experimental design was that the reporter gene construct (Tbait-hs-GFP) was targeted to be introduced between the transcription start site and the first methionine. This method has several advantages compared to conventional knock-out methods of introducing indels. First, the evaluation of sgRNAs can be achieved readily by screening reporter gene expression in injected animals. The time-consuming molecular biology steps, including DNA extraction, PCR reaction, and sequencing, are not needed. Strikingly, in the present study, we tried only one sgRNA for each of the genes (sox5, pax7a, and pnp4a) and obtained good results in all cases (Table 2). Second, upon establishment of mutant alleles, the strains express a reporter gene in the expression domains of the gene of interest. This makes it possible to monitor the morphological defects of the affected tissues by the expression of the reporter gene. Third, the presence of the reporter gene is greatly beneficial for selecting animals that will inherit the mutant allele in future generations.
Usage of the hsp70 promoter
As we did in zebrafish, we knocked in constructs with the hsp70 promoter (Figs. 1b and 4a). For this, we extracted a 0.8 kb sequence from the promoter of the medaka hsp70.1 gene . We showed in this study that this fragment works well as a basal (minimal) promoter. There are several advantages to utilizing the hsp70 promoter in DNA constructs for knock-in. First, the efficiencies of obtaining transgenic founders are likely to be increased, as reporter gene expression occurs irrespective of the direction of integration. Indeed, we found both types of integration (including multi-copy integrations) in the transgenic lines generated in this study. Second, the reporter construct can be targeted virtually anywhere near or within a gene of interest. This feature gives researchers flexibility in designing sgRNAs.
There are two potential disadvantages or concerns regarding the usage of the hsp70 promoter. First, the promoter has activity in female germ cells. We observed maternally derived fluorescence in many of the lines generated in this study. Although this did not present major problems in the present study, it could be a problem for monitoring the expression patterns of genes in the early stages. Nonetheless, this problem can likely be overcome, in many cases, by using transgenic males for mating. Second, gene expression may not be completely recapitulated with the usage of a heterologous promoter. Although reporter gene expressions appeared to mimic the expressions of the endogenous genes in the transgenic fish generated in the current study for all five of the genes, the possibility of an occurrence of ectopic expressions cannot be completely excluded. Indeed, we have noted an ectopic expression in a Tg[evx2-hs:Gal4] transgenic fish strain in the case of zebrafish . Thus, researchers need to be aware of the potential occurrence of an ectopic expression when using the hsp70 promoter.
Conversion of reporter gene expression by the Cre-loxP system
In this study, we showed that RFP expression can be changed to GFP expression in transgenic fish with the loxP-RFP-loxP-GFP construct. To make this occur ubiquitously, we established Tg[zhspa8:Cre-mCherry-NLS] in which the Cre-mCherry-NLS fusion protein is expressed ubiquitously in early embryos. Using this line, one reporter gene was easily converted to another reporter/driver gene by crossing alone. This is a powerful system for establishing transgenic fish that express a reporter/driver gene that is itself not fluorescent (without the aid of fluorescent reporters, prescreening before raising animals is not possible). For example, we succeeded in establishing several Gal4 driver lines using this system in zebrafish (our unpublished observation). In these studies, we first established loxP-RFP-loxP-Gal4 lines, and RFP was then converted to Gal4 by utilizing Tg[zhspa8:Cre-mCherry-NLS] transgenic zebrafish. The same strategy could be used to generate transgenic medaka that express reporter/driver genes that are not fluorescent themselves.
We report that the NHEJ-mediated knock-in system is highly efficient in medaka, and is very useful for establishing mutant alleles. With its simplicity and high efficiency, we propose that the method described may become a standard technique for the generation of transgenic and mutant medaka.
We thank the Medaka National Bioresource Project (NBRP Medaka), which is supported by the Japan Agency for Medical Research and Development, for providing the d-rR (MT837) and gu (MT827) strains. We also thank Ms. T. Yamazaki, H. Ito, and Y. Terasawa for their excellent technical assistance.
This work was supported in part by grants from the Ministry of Education, Science, Technology, Sports and Culture of Japan.
Availability of data and materials
Donor plasmids, as well as their sequence information, are available from the Medaka National Bioresource Project in Japan (NBRP Medaka).
SH conceived and designed the study. IW performed the major parts of the experiments. YK helped some of the experiments that IW performed. HH performed the experiments related to the sox5 and pax7a genes. SY and KN performed the experiments related to the pnp4a gene. All authors contributed to the writing of the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Fish were maintained and used in accordance with the guidelines approved by the animal care and use committees of the National Institutes of Natural Sciences (approval number: 17A002) and Nagoya University.
Consent for publication
The authors declare that they have no competing interests.
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