FIGURE SUMMARY
Title

Identification of maternal-effect genes in zebrafish using maternal crispants

Authors
Moravec, C.E., Voit, G.C., Otterlee, J., Pelegri, F.
Source
Full text @ Development

Maternal crispants can be used to efficiently identify maternal-effect genes in zebrafish in a single generation. (A) The traditional workflow for CRISPR-Cas9 in zebrafish uses one guide RNA that is co-injected with Cas9 mRNA or protein into the one-cell embryo to target a specific gene of interest. Successful targeting will result in adults with a mosaic germ line. To isolate these alleles, non-mosaic F1 fish are generated by crossing the F0 CRISPR-injected fish with a wild-type fish, then screening for heterozygous carriers of indels. Outcrossing followed by incrossing results in female homozygous mutants for a maternal-effect gene, phenotypes of which can be assessed in their offspring. (B) The maternal crispant technique generates maternal-effect phenotypes in a single generation. This strategy multiplexes four guide RNAs to target a single gene, thus increasing the rate of biallelic editing. If this biallelic editing event occurs in the germ line, the F1 offspring of F0 CRISPR-injected females have the potential to display a maternal-effect phenotype.

Identification of known maternal-effect genes using maternal crispants. (A-I) Known maternal-effect phenotypes resulting from the mutagenesis of three genes, birc5b (A,D,G), tmi (B,E,H) and mid1ip1l (C,F,I), were replicated using the maternal crispant technique. (A-C) Gene structure diagrams of birc5b (A), tmi (B) and mid1ip1l (C) showing the target sites of guide RNAs (red lines) and PAM sites (red stars). (D-F) Representative comparisons of a live wild-type (WT) embryo with live time-matched maternal crispant embryos of birc5b [D; 75 min post-fertilization (mpf)], tmi (E; 75 mpf) and midip1il (F; 150 mpf), showing defects in cytokinesis identical to previous studies. The mid1ip1l maternal crispant embryo shown exhibits a partially syncytial phenotype (box in F). (G-I) Immunohistochemistry labeling of β-catenin and DAPI staining in birc5b (G; 90 mpf), tmi (H; 90 mpf) and mid1ip1l (I; 75 mpf) maternal crispants showing the lack of accumulation of β-catenin in mature furrows, confirming a failure in furrow formation in spite of DNA replication. mid1ip1l maternal crispants contain ectopic β-catenin-containing vesicles near the cortex. In all immunohistochemistry labeling, insets show high magnification images of the boxed areas, highlighting the differences in β-catenin localization (arrowheads) and asymmetric DNA segregation (arrow). Note that there is some embryo size variation caused by methanol storage of embryos. Scale bars: 100 μm.

Gene structure and expression of kpna7 during development. (A) Expression levels of kpna7 in other species display a similar pattern of the maternal expression of kpna7 homologs. (B) Expression levels of the Kpna gene family throughout zebrafish development, from zygote to Prim-5 (24 hpf) stage. The maternal-specific transcript, kpna7, is represented in red and the gray bar marks the zygotic genome activation. (C) Diagram of the gene structure of kpna7, showing the known motifs and target sites of guide RNAs (red lines) and PAM sites (red stars).

Kpna7 is necessary for nuclear segregation during early development. (A) Images of live kpna7 maternal crispant and wild-type (WT) controls at the 8-cell stage (75 mpf) and shield stage (6 hpf). At the 8-cell stage, kpna7 maternal crispants appear to divide normally, but they stall at the sphere stage and fail to undergo epiboly. (B) Immunohistochemistry labeling of β-catenin and DAPI staining at 6 hpf showing that the kpna7 maternal crispant embryos exhibit nuclei of unequal sizes, including a subset of cells that entirely lack nuclei (blue asterisks). (C) Immunohistochemistry labeling of γ-tubulin and DAPI staining at 75 mpf, showing that kpna7 maternal crispant embryos display abnormal nuclear segregation (arrows) during cell cleavage. Scale bars: 100 μm (B,C, low magnification); 20 μm (B,C, high magnification).

Maternal expression and gene targeting of fhdc3 during development. (A) Expression levels of the Fhdc gene family throughout zebrafish development, from zygote to Prim-5 (24 hpf) stage. The maternal-specific transcript, fhdc3, is represented in red and the gray bar marks the zygotic genome activation. (B) Diagram of the gene structure of fhdc3, showing the known motifs and target sites of guide RNAs (red lines) and PAM sites (red stars). Expression data from early development in other systems is not yet known.

View largeDownload slide Fhdc3 is necessary to maintain the yolk-blastodisc boundary during early development. (A) Images of live embryos comparing fhdc3 maternal crispant embryos with time-matched wild-type (WT) controls during embryonic development. An abnormal constriction at the boundary between the blastodisc and yolk (black arrowhead) is observed during the early cleavage stages (64-cell). At later stages, a normal degree of constriction at the yolk-blastodisc boundary is restored, but embryos exhibit presumptive ectopic yolk inclusions (blue arrows). (B) At 1 dpf, fhdc3 maternal crispant embryos do not exhibit gross morphological defects, although they still contain presumptive ectopic yolk inclusions (arrows). (C) Immunolabeling of β-catenin and DAPI staining at 8-cell and 64-cell stages, showing that the overall shape and cell organization of the embryo are affected in the later fhdc3 maternal crispant embryos. (D) At the 8-cell stage, there is no significant difference in the shape of wild-type and fhdc3 maternal crispant embryos [fhdc3 maternal crispant score of circularity 0.881 (n=18); wild type 0.8615 (n=13); P=0.0893] but the shape appears significantly different at the 64-cell stage [fhdc3 maternal crispants: 0.90963 (n=8), wild type: 0.84757 (n=7); P=0.0003]. Error bars represent s.e.m. (E) At the 16-cell stage, the cortical F-actin ring appears significantly wider in the fhdc3 maternal crispant [fhdc3 maternal crispants: 24.8 μm (n=10), wild type: 14.1 μm (n=10); P=0.0003]. Error bars represent s.e.m. Brackets indicate the width of the cortical F-actin ring at the location shown in the schematic. Scale bars: 100 μm (C); 20 μm (E).

Indels can be identified in maternal crispants by producing haploids. IVF of crispant oocytes with UV-treated sperm generates embryos with a single maternal allele, facilitating Sanger sequencing and genetic analysis.

Sequencing data from haploid birc5b maternal crispants. (A) Chromatograms of sequences for guide sites that contained indels after CRISPR-Cas9 mutagenesis in a wild-type (WT) embryo and birc5b maternal crispant haploids from three different F0 fish. (B) Alignment of sequences corresponding to the chromatograms, with the guide sites in purple and the PAM sites in blue. Red text indicates the addition of new base pairs and dashes deleted bases.

Sequencing data from maternal crispant haploids for tmi, kpna7 and fhdc3 showing the generated lesions. (A-C) Alignment of sequences for tmi (A), kpna7 (B) and fhdc3 (C) maternal crispants with the guide sites in purple and the PAM sites in blue. Red text indicates the addition of new base pairs and dashes deleted bases.

Acknowledgments
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