Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish

Yizhuang Zhang; Ziping Fu; Boya Yang; Jiasheng Wang; Tong Lu; De-Li Shi; Ming Shao

doi:10.3791/68642

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Method Article

Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish

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DOI:

10.3791/68642

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July 22nd, 2025

Yizhuang Zhang¹ , Ziping Fu¹ , Boya Yang¹ , Jiasheng Wang¹ , Tong Lu¹ , De-Li Shi²^,³ , Ming Shao¹^,⁴^,⁵^,⁶

¹Shandong Provincial Key Laboratory of Animal Cell and Developmental Biology, School of Life Sciences Qilu Hospital (Qingdao), Cheeloo College of Medicine, Shandong University, ²Institut de Biologie Paris-Seine (IBPS), UMR CNRS 8263, INSERM U1345, Development, Adaptation and Ageing, Sorbonne Université, ³College of Marine Life Sciences, Ocean University of China, ⁴Key Laboratory for Experimental Teratology of the Ministry of Education, Shandong University, ⁵State Key Laboratory of Microbial Technology, Shandong University, ⁶Shandong University-Yuanchen Joint Biomedical Technology Laboratory

Summary

Here, we describe a protocol for generating maternal mutant that couples a stable zpc:cas9 knock-in line with Tol2-mediated delivery of sgRNA expression cassettes.

Abstract

Oogenesis and early embryonic development are critically dependent on maternally derived mRNAs and proteins. Eliminating these maternal factors necessitates homozygous mutations in female zebrafish, often resulting in lethal or infertile phenotypes, which prevent the acquisition of maternal mutant embryos. Our previous work introduced a rapid approach to bypass zygotic lethality through oocyte-specific genome editing. However, the previously reported cas9 transgene exhibits instability and undergoes gradual silencing over successive generations. Furthermore, the presence of Tol2 transposable elements flanking the zpc:cas9 cassette in this line hinders the potential for further sgRNA transgenesis using Tol2 system, which is currently the most efficient transgenic system in zebrafish. Consequently, there is a critical need for a Tol2-free zebrafish line that ensures stable and robust oocyte-specific Cas9 expression. Here, we present a line with zpccas9 knock-in at the rbm24a locus that addresses this requirement. Using this enhanced tool, we provide a pipeline for the rapid generation of maternal mutants of genes with zygotically lethal mutant phenotypes within the zebrafish model.

Introduction

The early embryonic development of vertebrates relies heavily on the RNA and proteins stored within the egg, collectively referred to as maternal factors. These maternal products are predominantly synthesized during the diplotene stage of meiosis I¹^,²^,³. To investigate their functional roles, it is essential to generate homozygous mutant female individuals, as only homozygous mutation in the oocytes can fully deplete these maternal factors. However, obtaining such homozygous mutant females becomes challenging when zygotic homozygous mutations result in lethality or sterility<....