A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish

Abstract

Maternal products are those mRNAs and proteins deposited during oogenesis, which play critical roles in controlling oocyte formation, fertilization, and early embryonic development. However, loss-of-function studies for these maternal factors are still lacking, mainly because of the prolonged period of transgenerational screening and technical barriers that prevent the generation of maternal (M) and maternal and zygotic (MZ) mutant embryos. By the transgenic expression of multiple sgRNAs targeting a single gene of interest in the background of a transgenic line Tg(zpc:zcas9) with oocyte-specific cas9 expression, we have successfully obtained maternal or maternal–zygotic mutant for single genes in F1 embryos. In this work, we tandemly connected a maternal GFP marker and eight sgRNA expression units to target dvl2 and dvl3a simultaneously and introduced this construct to the genome of Tg(zpc:zcas9) by meganuclease I-Sce I. As expected, we confirmed the existence of Mdvl2;Mdvl3a embryos with strong defective convergence and extension movement during gastrulation among outcrossed GFP positive F1 offspring. The MZdvl2;MZdvl3a embryos were also obtained by crossing the mutant carrying mosaic F0 female with dvl2+/−;dvl3a−/− male fish. This proof-of-principle thus highlights the potential of this conditional knockout strategy to circumvent the current difficulty in the study of genes with multiple functionally redundant paralogs.

Circumventing Zygotic Lethality to Generate Maternal Mutants in Zebrafish

“…In this situation, the mutations can be neither transferred to host embryos through germ-line replacement nor transmitted to the next generation through genetic mosaic females. Also of note, the oocyte expression of Cas9 and multiple gRNAs was found to induce large deletions in the genome with high efficiency compared with genome editing in fertilized eggs [ 22 , 23 ]. This may facilitate the analyses of promoter regions and transcriptional or post-transcriptional elements.…”

“…A novel strategy that allows for the generation of maternal or MZ mutants in the absence of viable and fertile homozygous mutant adults and can potentially be applied in the functional screening of maternal factors was developed recently [ 22 , 23 ]. It takes advantage of the established transgenic line Tg( zpc:zcas9 ) that allows the specific expression of zebrafish codon-optimized cas9 as early as in stage I oocytes under the control of the zona pellucida ( zpc ) gene promoter [ 31 , 32 ].…”

“…This oocyte-specific conditional knockout strategy is technically accessible and presents the advantage that founder fish can be used repeatedly in their lifespan. Moreover, it can be used to simultaneously inactivate multiple maternal genes with functional redundancy [ 23 ], although in this case, the editing efficiency needs further improvement.…”

“…Recently, a novel strategy has been developed for the generation of maternal mutants and maternal-zygotic (MZ) mutants by circumventing zygotic lethality in a technically accessible, time-saving, and efficient manner [ 22 , 23 ]. It relies on the oocyte transgenic expression of CRISPR/Cas9 and guide RNAs (gRNAs) to target genes of interest and produces maternal mutants in the next generation (3 months).…”

Zebrafish as a Model to Study Vascular Elastic Fibers and Associated Pathologies

“…The CRISPR technique has also been developed to perform knockins and conditional knockouts in the zebrafish [15,16]. These modifications can be helpful to implement models with relevant characteristics of human diseases, such as specific variants associated with pathologies [17], restriction of genetic modifications to a precise time period and/or location [18], and also to avoid the compensations that are often associated with knockout experiments [19].…”