Highly efficient genome editing using oocyte-specific zcas9 transgenic zebrafish

Abstract

Highly efficient genome editing using oocyte-specific zcas9 transgenic zebrafish Since its first application to induce mutations in mammalian cells (Cong et al., 2013; Mali et al., 2013), CRISPR/Cas9 rapidly becomes a routine technique to perform genome editing in a variety of biological systems due to its facile, robust, and multiplexable features (Hwang et al., 2013; Guo et al., 2014; Wang et al., 2013). In every system, Cas9 and sgRNA should co-express to induce mutations. This is typically achieved by injection of the sgRNA mixed with Cas9 mRNA or protein into the fertilized eggs in zebrafish. As the in vivo translation of capped RNAs delay the presence of sufficient Cas9 protein, the mRNA injection produces more mosaic animals and shows relatively lower efficiency when compared to Cas9 protein injection (Burger et al., 2016). However, it is much more cost effective than using commercially available Cas9 protein, especially in the case of large-scale mutation screens. To further simplify this procedure, lower the cost and maintain an acceptable high genome editing efficiency in zebrafish, we set out to generate a transgenic line with stable and specific Cas9 expression in the egg. zpc (zp3b) promoter (zpc0.5) was reported to drive robust and specific GFP expression in the oocyte (Onichtchouk et al., 2003), we thus fused it with a zebrafish codon optimized cas9 (zcas9) followed by an SV40 3' UTR (Fig. 1A and File S1; Liu et al., 2014), and introduced this construct to zebrafish genome by Tol2 mediated transgenesis (Urasaki et al., 2006). To screen female F0 fish harboring this transgene, the F0 female founders were outcrossed with the wild-type male, the resulting eggs were injected with an sgRNA targeting slc45a2 (Moreno-Mateos et al., 2015), a gene essential for melanin synthesis, and assayed for their pigmentation phenotype. We found that the offspring of one founder showed a high proportion of pigmentation defect. Some of them exhibited a homozygous mutant like albino phenotype, suggesting a robust Cas9 expression in the egg and a very high efficient gene disruption. To examine the specificity of zcas9 expression in the transgenic line, the offspring (F1) of this F0 founder were raised to adulthood, and the ovaries of female F1 transgenic fish were dissected and subjected to in situ hybridization (ISH) employing the probe of zcas9. We found that the transcripts of zcas9 were specifically distributed in oocytes with a diameter of 40-200 µm, at around IB-II stages, while in WT embryos, no hybridization signals were detected (Fig. 1B, C). We did not detect zcas9 expression in stage IA oocytes or in larger ones after stage II. Accordingly, by RT-PCR, we also failed