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. 2021 Aug 16;10(8):777. doi: 10.3390/biology10080777

Figure 1.

Figure 1

Workflow to generate double maternal mutants for dvl2 and dvl3a genes. (A) Two cloning steps to generate transgenic vectors containing eight sgRNA expression cassettes. By Golden Gate ligation, four sgRNA expression cassettes are cloned tandemly into pGGDestISceIEG-4sgRNA backbone, generating pGGDestISceIEG-4sgRNA (dvl2) and pGGDestISceIEG-4sgRNA (dvl3a). The four sgRNAs targeting dvl3a were then amplified from the pGGDestISceIEG-4sgRNA (dvl3a) plasmid and inserted into the Acc65I site in pGGDestISceIEG-4sgRNA (dvl2) vector via T5 exonuclease-dependent assembly (TEDA). (B) The resultant pGGDestISceIEG-8sgRNA (dvl2;dvl3a) was coinjected with I-Sce I into embryos spawned by a wild-type female and a Tg(zpc:zcas9) homozygous male. The mosaic female with germ-line transmission was outcrossed to produce GFP-positive embryos, which were then genotyped and phenotyped for double maternal mutations.