CRISPR/Cas9 genome editing is revolutionizing genetic loss-of-function analysis but technical limitations remain that slow progress when creating mutant lines. First, in conventional genetic breeding schemes, mosaic founder animals carrying mutant alleles are outcrossed to produce F1 heterozygotes. Phenotypic analysis occurs in the F2 generation following F1 intercrosses. Thus, mutant analyses will require multi-generational studies. Second, when targeting essential genes, efficient mutagenesis of founders is often lethal, preventing the acquisition of mature animals. Reducing mutagenesis levels may improve founder survival, but results in lower, more variable rates of germline transmission. Therefore, an efficient approach to study lethal mutations would be useful. To overcome these shortfalls, we introduce 'leapfrogging', a method combining efficient CRISPR mutagenesis with transplantation of mutated primordial germ cells into a wild-type host. Tested using Xenopus tropicalis, we show that founders containing transplants transmit mutant alleles with high efficiency. F1 offspring from intercrosses between F0 animals that carry embryonic lethal alleles recapitulate loss-of-function phenotypes, circumventing an entire generation of breeding. We anticipate that leapfrogging will be transferable to other species.
Pubmed ID: 27385011 RIS Download
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Supports researchers using Xenopus models. Researchers are encouraged to deposit Xenopus transgenic and mutant lines, Xenopus in situ hybridization probes, Xenopus specific antibodies and Xenopus expression clones with the Centre. EXRC staff perform quality assurance testing on these reagents and then make them available to researchers at cost. Supplies wild-type Xenopus, embryos, oocytes and Xenopus tropicalis fosmids.
View all literature mentionsData collection for Xenopus laevis and Xenopus tropicalis biology and genomics.
View all literature mentionsSupports researchers using Xenopus models. Researchers are encouraged to deposit Xenopus transgenic and mutant lines, Xenopus in situ hybridization probes, Xenopus specific antibodies and Xenopus expression clones with the Centre. EXRC staff perform quality assurance testing on these reagents and then make them available to researchers at cost. Supplies wild-type Xenopus, embryos, oocytes and Xenopus tropicalis fosmids.
View all literature mentionsData collection for Xenopus laevis and Xenopus tropicalis biology and genomics.
View all literature mentions