Materials and Reagents

Plasmid pDR274 (Addgene, catalog number: 42250 )
pBaitD-gap43-linker-EGFP (RIKEN DNA BANK, catalog number: RDB15409 )
Ampliscribe T7-Flash Transcription Kit (Epicentre, catalog number: ASF3257 )
RNeasy Plus Mini Kit (QIAGEN, catalog number: 74134 )
PCR-Kit; KOD-Plus-Neo (TOYOBO, catalog number: KOD-401 )
Sodium hydroxide (NaOH) (NACALAI TESQUE, catalog number: 31511-05 )
Ethylenediaminetetraacetate acid (EDTA) (NACALAI TESQUE, catalog number: 14347-21 )
Tris-HCl (pH 8.0) (NACALAI TESQUE, catalog number: 35435-11 )
Alkaline lysis buffer (see Recipes)
Neutralization buffer (see Recipes)

Equipment

Forceps (DUMONT, model: 91-3869 or equivalents)
PCR thermal cycler (NIPPON Genetics, catalog number: TC-96GHbC )
Incubator (NKsystem, catalog number: LH-60FL3-DT )
Fluorescent microscope (Leica, catalog number: Leica MZ8 )

Procedure

Search target sequences for gRNAs using a gRNA-scoring algorithm (CRISPRscan; Moreno-Mateos et al., 2015), and select the one that has less off-target sites in medaka genome.
Obtain oligonucleotides for the selected gRNA from a supplier of choice.
Anneal the oligonucleotides and insert the annealed fragment into gRNA expression plasmid pDR274 (Addgene Plasmid #42250).
Transcribe from the DraI-digested plasmid using the Ampliscribe T7-Flash Transcription Kit, and purify the transcript with the RNeasy Plus Mini Kit to eliminate the template DNA without DNase treatment.
PCR-amplify the upstream and downstream regions (homology arms: ca. 500 bp) of the genomic target site using the region-specific primers with restriction enzyme sites, which help to insert into the donor plasmid described in Step 6.
Ligate both the homology arms, the insert fragment containing a linker, a reporter gene, and a polyA signal into the backbone plasmid containing the BaitD sequence that helps to induce gene knock-in events with high efficiency in medaka (Figure 1A).
Note: The donor plasmid containing BaitD can be obtained from RIKEN DNA BANK (pBaitD-gap43-linker-EGFP #15409). The sequence of the donor plasmid is shown as Supplemental file 1.

Figure 1. Schematic illustration of a gene knock-in experiment. A. Schematic design of the donor plasmid. The left (blue in (A)) and right (green in (A)) homology arms (ca. 500 bp) are amplified respectively using the region-specific primers with restriction enzyme sites (XhoI/BamHI or EcoRI/SpeI). Linker sequence stabilizes to express the reporter gene (GFP or RFP) as a fusion protein with the target gene product. Bait is a sequence to be cleaved for linearization of the donor plasmid, and enhances gene knock-in events in medaka. Thunder marks show the cleavage sites on the genome and the donor plasmid by Cas9 and gRNA1 or gRNA2, respectively. B. Schematic illustration showing precise integration of the insert gene into the genomic target site. The reporter gene (GFP or RFP) is integrated into exon and in-frame. Blue and green triangles show the junctions between the genomic target site and the fragment of donor plasmid. Primer pairs labeled (1)/(2) or (3)/(4) can be used for amplifying the upstream or downstream junction region. To confirm the precise integration into the genomic target site, primer (1) and (4) are designed on the outside of the junctions.
Inject the following solution into embryos; 100 ng/µl of Cas9 RNA, 50 ng/µl of sgRNAs for cleaving the genomic target site and the donor plasmid, and 2.5 ng/µl of each donor plasmid.
Note: Please refer to ‘Medaka-microinjection’ for the details of microinjection protocol.
Raise the embryos expressing each reporter gene to adult for about 2 months.
Mate with wild type counterparts. (First mating)
Extract genomic DNA from the resultant F1 embryos expressing each reporter gene, according to the following protocol:
1. Put embryos in 25 µl of alkaline lysis buffer (see Recipes).
2. Incubate at 95 °C for 15 min after breaking the egg envelope with forceps.
3. Neutralize with 25 µl of neutralized buffer (see Recipes).
Note: DNA extraction is only necessary for F1 generation, because it is needed to investigate by the sequence analysis whether the insert gene is correctly integrated into the genomic target site.
PCR-amplify the junction regions of the target site on the host genome and the introduced gene using F1 genomic DNA (Figure 1B).
Note: The PCR condition is performed as described in the protocol of KOD-Plus-Neo (Toyobo).
Perform the sequence analysis of the resultant PCR amplicons, to confirm the precise integration into the genomic target site.
After identifying F0 founders with the desired mutation, mate with wild type counterparts again. (Second mating)
Note: First mating is for the identification of F0 founders harboring the insert gene in germ cells, while second mating is for the establishment of gene knock-in strains in F1 generation.
Collect F1 embryos expressing each reporter gene, and raise to fish with a length > 1 cm.
Cut off tail fin from each F1 fish, and perform the genome extraction and the sequence analysis described as above.
After confirming the precise integration into the genomic target site in each F1 fish by the sequence analysis, mate F1 fish harboring different color reporter genes with each other.
Observe the fluorescence color of the resultant F2 embryos, to specify the individuals integrated the insert gene on the target site.
Note: The resultant F2 embryos are genotyped alive by fluorescence color: wild type without fluorescence, monoallelic mutants for integration with one fluorescence, and biallelic mutants for integration with both fluorescence.

Recipes

Alkaline lysis buffer
25 mM NaOH
0.2 mM EDTA
Neutralization buffer
40 mM Tris-HCl (pH 8.0)

Acknowledgments

This protocol was adapted from our previous works (Murakami et al., 2017). The work was partially supported by a Grant-in-Aid for Scientific Research (A) 15H02540 (MK) and a Grant-in-Aid for JSPS fellows 13J01682 (SA). The funders had no role in the design of the study and collection, analysis, or interpretation of data or in the writing of the manuscript. The authors declare no conflicts of interest or competing interests with this manuscript.

References

Fujimori, K. E., Kawasaki, T., Deguchi, T. and Yuba, S. (2008). Characterization of a nervous system-specific promoter for growth-associated protein 43 gene in Medaka (Oryzias latipes). Brain Res 1245: 1-15.
Moreno-Mateos, M. A., Vejnar, C. E., Beaudoin, J. D., Fernandez, J. P., Mis, E. K., Khokha, M. K. and Giraldez, A. J. (2015). CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo. Nat Methods 12(10): 982-988.
Murakami, Y., Ansai, S., Yonemura, A. and Kinoshita, M. (2017). An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes). Zoological Lett 3: 10.