Highly Efficient Genome Editing Using Oocyte-specific Zcas9 Transgenic Zebrafish

For almost all vertebrates,transcription is not activated at the onset of the development until the Mid-Blastula Transition(MBT),which means that early embryogenesis is heavily dependent on maternally supplied mRNAs and Proteins.These maternal factors can also exert their influence long after the zygotic genome activation,compensating the function of zygotic products.The formation of embryonic axis,morphogenetic movements and the determination of primordial germ cells are mainly controlled by maternal factors.However,as lacking a simple and efficient strategy to eliminate maternal gene products,it is still difficult to perform mechanistic studies on maternal factors and their associated developmental processes.Using Tol2 transposon system and the zpc promoter to drive the expression of a zebrafish codon optimized cas9(zcas9),we have generated a transgenic line,Tg(zpc:zcas9htgh),with stable and specific Cas9 expression in the egg.This line maintains a comparable editing efficiency with that of the Cas9 mRNA or protein injections and thus can be used to perform conventional gene knockouts.The results of qPCR and Southern blotting analysis showed that only single copy of cas9 expression cassette inserted to the genome.And then the insertion site was further mapped to the third intron of gfra2a gene by inverse PCR.This insertion,however,does not affect the expression level and pattern of gfra2a,as well as the quality,quantity and survival of the transgenic offspring.Next,an sgRNA expression vector with GFP selection marker was introduced to the genome of Tg(zpc:zcas9high)by I-SceI mediated transgenesis to target the maternal expression of nanog.We found that 15.9%of GFP positive embryos exhibited a severely delayed epiboly movement,phenocopying the maternal mutant of nanog previously reported.In summary,we have created a transgenic line with oocyte specific cas9 expression.As a tool,it can be used not only in classic genome editing,but also in the application to manipulate maternal factors.It can significantly simplify the workflow and lowers the cost of conventional gene knockout procedure in zebrafish.Besides,our "proof of principle"experiment also verified its feasibility to eliminate maternal gene products,thus making this strategy potentially useful for the investigation of oogenesis and early developmental processes.