Rapid generation of maternal mutants via oocyte transgenic expression of CRISPR-Cas9 and sgRNAs in zebrafish

Abstract

Maternal products are exclusive factors to drive oogenesis and early embryonic development. As disrupting maternal gene functions is either time-consuming or technically challenging, early developmental programs regulated by maternal factors remain mostly elusive. We provide a transgenic approach to inactivate maternal genes in zebrafish primary oocytes. By introducing three tandem single guide RNA (sgRNA) expression cassettes and a green fluorescent protein (GFP) reporter into Tg(zpc:zcas9) embryos, we efficiently obtained maternal nanog and ctnnb2 mutants among GFP-positive F1 offspring. Notably, most of these maternal mutants displayed either sgRNA site–spanning genomic deletions or unintended large deletions extending distantly from the sgRNA targets, suggesting a prominent deletion-prone tendency of genome editing in the oocyte. Thus, our method allows maternal gene knockout in the absence of viable and fertile homozygous mutant adults. This approach is particularly time-saving and can be applied for functional screening of maternal factors and generating genomic deletions in zebrafish.

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

“…Intriguingly, all these amplicons were wild-type as determined by sequencing. This unusual phenomenon reminded us of the deletion-prone tendency in creating nanog and ctnnb2 maternal mutants [ 18 ]. Since these defective embryos were generated after crossing a GFP-positive F0 female with a wild-type male, some of the eight sgRNA targets must be in a heterozygous mutant status.…”

“…In this work, we observed 5.4% of embryos were M dvl2 ;M dvl3a among GFP-positive sgRNA expressing embryos. This is predictable, because we obtained an average of 25–38% maternal mutants for a single gene using this system [ 18 ]. So theoretically, the ratio for double maternal mutant should be below 10% when calculating the square of the mutant proportions for single genes.…”

“…Wild type AB and Tg( zpc : zcas9 ) were maintained in standard conditions. The Tg( zpc : zcas9 ) was previously described [ 18 ] and is available in China Zebrafish Resource Center (CZRC). Embryos were raised at 28.5 °C.…”

“…In our recent work, we efficiently generated nanog and ctnnb2 maternal mutants in just one fish generation by introducing sgRNA expression cassettes in the background of Tg( zpc : zcas9 ), which expresses zebrafish codon-optimized Cas9 under the control of the zona pellucida gene zp3b [ 18 , 19 ]. Compared to previous methods, this novel oocyte-specific conditional knockout (CKO) strategy presents several advantages.…”

“…First, it is much more time-saving, as the time to get M or MZ mutants can be reduced to about three months. Second, this method is also efficient in generating maternal mutants of single genes; after expressing three sgRNAs targeting a gene of interest, more than 25% of GFP-positive embryos are expected to be the maternal mutant [ 18 ]. Notedly, this transgenic expression of multiple Cas9 RNPs in the oocyte can generate large deletions on the genes of interest with high efficiency and thus can theoretically prevent potential genetic compensation [ 18 ].…”