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

• Main Authors: Chong Zhang, Jiaguang Li, Imran Tarique, Yizhuang Zhang, Tong Lu, Jiasheng Wang, Aijun Chen, Fenfen Wen, Zhuoyu Zhang, Yanjun Zhang, Ming Shao
• Format: Article
• Language: English
• Published: MDPI AG 2021-08-01
• Series: Biology
• Subjects: maternal factors; oocyte; CRISPR/Cas9; double mutant; early development; zebrafish
• Online Access: https://www.mdpi.com/2079-7737/10/8/777

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(<i>zpc</i>:<i>zcas9</i>) with oocyte-specific <i>cas9</i> 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 <i>dvl2</i> and <i>dvl3a</i> simultaneously and introduced this construct to the genome of Tg(<i>zpc</i>:<i>zcas9</i>) by meganuclease I-<i>Sce</i> I. As expected, we confirmed the existence of M<i>dvl2</i>;M<i>dvl3a</i> embryos with strong defective convergence and extension movement during gastrulation among outcrossed GFP positive F1 offspring. The MZ<i>dvl2</i>;MZ<i>dvl3a</i> embryos were also obtained by crossing the mutant carrying mosaic F0 female with <i>dvl2</i>^+/−;<i>dvl3a</i>^−/− 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.