In our method, Cas9-gRNA is specifically expressed in germl cells to induce germline mutations.
For each gene, a transgenic line ubiquitously expressing a gRNA from the U6 promoter is established.
The U6-gRNA line is crossed to the nos-Cas9 line, which expresses Cas9 specifically in germ cells.
Progeny of the cross carry both the nos-Cas9 and U6-gRNA transgenes, which leads to formatin of an active Cas9-gRNA complex specifically in germ cells.
Male founder animals expressing Cas9-gRNA are crossed to wild-type females and their offspring are screend for an induced mutation by molecular genotyping.