Fig 1.
Design of gRNAs for the knock-out at the Dma-ey locus.
This figure was drawn based on a previously published schematic illustration [25]. The arrowheads indicate the recognition sites of gRNA-1 and gRNA-2. An arrow indicates the target site of TALEN as previously reported [26].
Fig 2.
Phenotype of Dma-ey knock-out.
The left image shows a normal compound eye (ce) in the wild type (WT) strain. The right image shows the Dma-ey knock-out phenotype with a deformed compound eye. The scale bar indicates 100 μm.
Table 1.
Summary of knock-out experiments.
Fig 3.
Structure of a donor plasmid for the knock-in at the Dma-ey locus.
The Thosea asigna virus 2A (T2A) peptide connected the mCherry marker with a fusion protein comprising histone 2B (H2B) connected to green fluorescent protein. The ef1a1 promoter/enhancer was inserted upstream from the T2A reporter cassette. The Cas9 target site (gRNA-2 recognition site) was derived from Dma-ey exon 10.
Fig 4.
Phenotype of transgenic D. magna.
The top row shows embryos obtained from wild type (WT) and transgenic (TG) lines. The middle and bottom rows show mature TG and WT D. magna, respectively. The images in each column were taken from the same individual under brightfield microscopy (left), with an mCherry filter (middle), and with a GFP2 filter (right). The regions surrounded by the white dashed lines are ovaries. The TG D. magna showed both red and green fluorescence. The mCherry fluorescence seen in the gut of the WT individuals is due to the autofluorescence of algae (chlorophyll has a fluorescence spectrum similar to that of mCherry and the gut is full of ingested algae). Under the GFP2 filter, which is a long pass filter with a peak transmission of 510 nm for emission, yellow autofluorescence could sometimes be detected, which was probably dependent on the gut contents as observed in the WT animals.
Table 2.
Summary of knock-in experiment.
Fig 5.
Characterization of the transgene structure.
(a) Illustration of the endogenous locus where donor DNA was integrated. rev2, fwd, 1F, 3R, 10F, and mid_R indicate the positions of primers used for the amplification of the regions A, B, and C by polymerase chain reaction to determine the structure of the integrated donor DNA. The pink, orange, green, grey, and blue boxes and the grey arrows indicate the mCherry coding region, T2A region, H2B-GFP coding region, ef1a1 3′ UTR, Cas9 target region, and ef1a1 promoter/enhancer region, respectively. (b) Polymerase chain reaction to determine the transgene structure. The amplified genomic DNA fragments were resolved by agarose gel electrophoresis. (c) The sequences of the junction regions between the transgene and the surrounding genome. The target sites for gRNA are indicated by blue letters and insertions are indicated by red letters. The protospacer adjacent motif sequence is underlined with red.