Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes

Abstract

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.

Keywords: CRISPR/Cas9; Genome editing; Knockouts; Primordial germ cells; TALENs; Xenopus.

Fig. 1.

Transplantation of PGCs. (A) Scheme for transplanting PGCs from CRISPR/Cas9-mutagenized blastula stage embryos (bottom) into a wild-type soma (top) that has had its PGCs removed. (B) Wild-type blastula showing vegetal localization of PGCs as detected by dazl in situ hybridization. (C) PGC explants show many foci of dazl expression. (D) Carcasses from blastula embryos show vastly reduced dazl expression foci, suggesting effective removal of PGCs.

Fig. 2.

Test crosses between animals carrying tyr-mutated leapfrog transplants and albinos demonstrate germline transmission of mutant alleles. (A) Leapfrog transplant-bearing male (pigmented) is shown amplexed with an albino tyr^−/− female. (B) Leapfrog transplant-bearing female (pigmented) is shown amplexed with an albino tyr^−/− male. (C) Examples of F1 progeny from the cross in A grown to tadpole stage. These tadpoles are albino because they inherited tyr mutant alleles from both F0 parents. Therefore the leapfrog-generated frog carries gametes derived from CRISPR-mutated PGCs. The inset in C shows an unrelated pigmented tadpole at roughly the same stage for comparison.

Fig. 3.

Whole-animal targeting of gsc causes a dramatic reduction in survival in F0 embryos. (A) The gsc gene structure is shown. The open reading frame (ORF) is shown in blue and the homeobox, in red, is split between exons 2 and 3, with the DNA recognition helix (VWFKNRR) coding sequence found downstream of the exon 3 splice acceptor. The CRISPR/Cas9 target site location is indicated. (B) Representative wild-type (uninjected) tadpole and (C) a gsc CRISPR-injected cyclopic tadpole at 9 dpf illustrate the extent of defects in head/craniofacial development. Insets show whole tadpoles. Tadpoles are shown at the same magnification, as are insets. (D) A survival curve shows that the population of gsc targeted F0 embryos is severely reduced by 14 dpf. Plots for uninjected siblings (Un) and tyr CRISPR-injected embryos are shown as controls. Equivalent amounts of gsc and tyr sgRNAs were used.

Fig. 4.

F1 embryos derived from intercrosses of F0 gsc leapfrogged adults show variable loss of anterior head structures. (A-C) Whole-mount in situ hybridization of F0 embryos using a cocktail of riboprobes for otx2, egr2 and hoxb9 marking increasingly posterior domains of the embryo. Loss of the anterior portion of the otx2 expression domain is seen (note region marked with asterisk in B that is not readily distinguishable in the embryo in C), while the more posterior expression domains remain unaffected. (D-F) From a second mating, 370 embryos were grown to mid-tailbud stage 40 to assess the severity of loss of anterior head structures. In approximately one-third of the embryos (E), eyes fuse in the anterior midline whereas in another third (F) a more severe anterior truncation is seen and eye structures fail to form.