Customization of a DR-GFP reporter
To visualize the HDR by fluorescence in D. magna, a previously established DR-GFP reporter plasmid20 was customized (Fig. 1A, B). A red fluorescent protein gene mCherry ORF was fused upstream of the SceGFP via a sequence encoding Thosea asigna virus 2A (T2A), which can lead to bicistronic expression of both mCherry and mutated/repaired eGFP proteins37. To distinguish the eGFP-expressing cells individually, the nuclear localization signal was included in the SceGFP. At 6 bp upstream of the I-SceI recognition site, we also introduced a recognition site (5′ -TCGA- 3′) of a four-base endonuclease TaqI to allow us for visualizing HDR not only by the targeted DSB with I-SceI but also by the multiple DSBs with TaqI on the genome38. DR-GFP reporter will function when the DSB is introduced in the I-SceI site by the Cas9-gRNA complex. By SDSA or non-crossover DSBR subpathway of HDR, SceGFP will use iGFP as a repair template resulting in the functional eGFP expression (Fig. 1C). To express the DR-GFP reporter, we chose a 2.3 kb of D. magna EF1α-1 genomic fragment including the promoter/enhancer, the transcription start site, the complete first intron, and part of the second exon with a start codon39. In addition, to recapitulate EF1α-1 endogenous expression, the full-length EF1α-1 3′ UTR was added downstream of the reporter. The complete nucleotide sequence of the customized DR-GFP reporter and the deduced amino sequence is provided in Supplementary Figure S1.
Generation of HDR reporter transgenic Daphnia
To integrate the customized DR-GFP reporter into D. magna genome, the CRISPR/Cas-mediated knock-in via non-homologous end-joining was used29. As a target site for knock-in of the reporter plasmid, we chose exon 3 of the eye pigment transporter scarlet gene33. We co-injected 50 ng/µL reporter plasmid and the RNP complex into 29 eggs. The 10 injected embryos survived until adult, from which 9 produced offspring with a white eye that is the typical phenotype of the scarlet mutant, indicating that the Cas9 RNP induced DSBs at the targeted site. Of the 9, one adult produced offspring with ubiquitous mCherry fluorescence, suggesting germline transmission of reporter plasmid (Fig. 2A). This fluorescence pattern also indicates that this reporter system enables us to detect the HDR event in most type of cells. We cultured this potentially transgenic line for genotyping.
To investigate whether NHEJ-mediated knock-in occurred, genotyping was performed using the genome of the potentially transgenic line. We amplified the mCherry fragment, 5′ and 3′ junctions between the transgene and its surrounding region by PCR (Fig. 2B). The expected size of the mCherry fragments was obtained only in the potential transgenic line (Fig. 2C, fragment B, DR-GFP line). The 3′ junction region was also amplified in this line by using forward primer targeted at the downstream of EF1α-1 3′ UTR of the donor plasmid and reverse primer targeted exon 8 of scarlet gene locus (Fig. 2B; Fig. 2C, fragment C, DR-GFP line). Sequencing of this PCR product confirmed the integration of the reporter plasmid at the scarlet locus and revealed 20 bp deletion and 8 bp insertion at the 3′ side of the integrated cassette (Fig. 2D, 3´ junction). Consistent with the white-eyed phenotype, another allele contained indel mutation at the DSB site (Fig. 2D, 2nd allele). We were unable to amplify the 5′ junction region even if the forward primer was designed at 3,157 bp upstream and 2,610 bp downstream of the DSB site. This suggests that large deletion occurred at the 5′ side of the integration site. Nevertheless, amplification and sequencing of the full-length of the DR-GFP gene cassette demonstrated the integration of the intact DR-GFP reporter (Supplementary Figure S1). We then named this knock-in daphniid the DR-GFP line.
The DSB near the I-SceI site leads to the generation of eGFP-positive cells in embryos of the DR-GFP line
To determine whether DSBs induce HDR within the DR-GFP reporter system, we attempted to introduce DSBs with Cas9 and gRNA named SceI gRNA that target the SceGFP. The DSB by this Cas9 RNP occurs 2 bp upstream of the I-SceI recognition site that was previously used for I-SceI-dependent DSBs20. To confirm whether Cas9 is active during microinjection, we planned to co-inject the SceI gRNA with another gRNA targeting Distal-less (Dll) gene. This is because knockdown of Dll in embryos of D. magna led to a distinct phenotype “truncation of second antennae” and the level of this truncation corresponded to the degree of impairment of this gene35. The Dll gRNA was targeted to the upstream of the homeodomain region in exon 2 (Supplementary Figure S2), as this region is highly conserved among arthropod35 and considered important for Dll function40,41. Injection of Cas9 and Dll gRNA into D. magna eggs resulted in 100% (13/13) truncation of second antennae (Supplementary Table S1). Of these, 8 (61.5%) showed the same strong phenotype as that induced by Dll RNAi35. This result suggests that Dll gRNA can be used as a marker for Cas9 activity during microinjection.
Seventy-four eggs were co-injected with 1 µM Cas9 protein and gRNA mixtures (SceI gRNA and Dll gRNA, 2 µM each). The phenotypes of the second antennae of the injected embryos were observed 48 hours post-injection (hpi). Forty-three embryos survived until the 48 hpi stage and 41 (95%) showed truncation of the second antennae (Table 3) from which, 22 embryos (54%) showed the strong phenotype35, indicating that Cas9 was active during injection and could introduce DSBs on the genome. Of the 41, 33 (80%) showed strong nuclear-localized eGFP fluorescence in the tissues such as the head and thoracic appendages. (Fig. 3). In contrast, embryos injected with Cas9 RNP including the unrelated St gRNA (Fig. 3) and Dll gRNA did not show intense and nuclear-localized GFP signal, indicating that the recovery of the eGFP fluorescence occurred by injection of Cas9 protein and SceI gRNA.
Table 3
Summary of Cas9 protein, SceI gRNA, and Dll gRNA co-injection
Injected
|
Developed
(48hpi)
|
Truncated antennae
|
Nuclear-localized eGFP
|
Strong
|
Medium
|
Mild
|
74
|
43
|
22/41 (54%)
|
9/41 (22%)
|
10/41 (24%)
|
33/41 (80%)
|
The embryos showing the nuclear-localized fluorescence signals have a functional eGFP gene repaired by HDR
To confirm whether HDR occurred at the genomic level, we extracted genome DNA from uninjected embryos and injected embryos that showed nuclear-localized eGFP fluorescence. PCR was then performed with a forward primer in the mCherry region and a reverse primer that recognizes specifically the sequence of the repaired SceGFP and (Fig. 4A, Fig. 4B). Because the reverse primer also can bind to the iGFP sequence that is a template for HDR (Fig. 4A), we expected two bands would appear upon genomic PCR. A higher size band (2,843 bp) was present in all samples, indicating amplification from iGFP sequence (Fig. 4C, ii), while a lower size band (1,048 bp) indicating amplification from repaired SceGFP sequence was obtained only from embryos injected with Cas9 and SceI gRNA (Fig. 4C, i). These results also suggest the repair of SceGFP by Cas9 and SceI gRNA.
We also attempted to develop a qPCR-based method that can detect the repaired SceGFP expression. We designed a forward primer that binds to the T2A-coding sequence of DR-GFP reporter locus, and a reverse primer that specifically binds to repaired SceGFP sequence (Fig. 5A). As a model to test this system, we used Cas9-mRNA and Cas9 protein for introducing the DSB on the SceGFP because mutagenesis efficiency with Cas9 mRNA was lower than that with Cas9 protein29, which suggested Cas9 mRNA induces DSB occurrence to a lesser extent. We introduced the DSB at the SceGFP following either optimum condition of Cas9 mRNA (500 ng/µL Cas9 mRNA and 50 ng/µL gRNA) or Cas9 protein injection (1 µM Cas9 protein and 2 µM gRNA) 34,29. The Dll gRNA was co-injected to evaluate the Cas9 activity in each injection. We confirmed 54% of Cas9 protein injected embryos showed a strong phenotype of second antennae truncation while Cas9 mRNA could only introduce a mild phenotype (Table 3 and Table 4). This result implied that Cas9 protein had stronger activity to introduce DSB. Subsequently, the level of repaired SceGFP was analyzed using qPCR. By Cas9 protein injection, we observed significantly higher expression of repaired SceGFP (~ 5 fold) relative to Cas9 mRNA injection. Moreover, neither repaired SceGFP signal nor amplification was detected in uninjected embryos as well as scarlet gRNA injected embryos (Fig. 5B, Supplementary Fig. 3). Our result shows that qPCR can be used to detect the functional eGFP repaired by HDR.
Table 4
Summary of Cas9 mRNA, SceI gRNA, and Dll gRNA co-injection
Injected
|
Developed
(48hpi)
|
Truncated antennae
|
Nuclear-localized eGFP
|
Strong
|
Medium
|
Mild
|
24
|
10
|
0
|
0
|
8/8 (100%)
|
Not observed*
|
*To confirm the integrity of the Cas9 mRNA, the eGFP mRNA was also co-injected for confirmation of the mRNA integrity based on the eGFP fluorescence intensity. This prevented us from observing the nuclear-localized eGFP signals in the Cas9 mRNA-injected embryos. |