Blitz IL et al. (2016), Leapfrogging: primordial germ cell transplantat...

XB-ART-52321

Development January 1, 2016; 143 (15): 2868-75.

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

Blitz IL , Fish MB , Cho KW .

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.

PubMed ID: 27385011
PMC ID: PMC5004912
Article link: Development
Grant support: [+]
Genes referenced: dazl dsp gsc pgc tyr
GO keywords: germ cell development

Article Images: [+] show captions

	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. S1. Assessment of mutagenesis on vegetal PGC-containing explants relative to the rest of the embryo. (A) Dissection scheme. Embryos were injected animally at the 1-cell stage with tyr sgRNA-complexed Cas9 protein. At blastula stage 9 they were dissected into animal cap and vegetal PGC transplant regions, and the embryo remainder composed of vegetal and marginal zone tissues (mesoderm and endoderm; Meso/endo). Individual embryo fragments were lysed and the tyr target region was PCR amplified. The amplicon population was subjected to direct sequencing (DSP; Nakayama et al., 2014). (B) DSP assay results are shown for each region from a single embryo. The position of the predicted Cas9 cleavage site is shown as a vertical line. One can see that, in comparison to the sequence profile of an uninjected embryo at top, all three regions from one injected embryo show similar profiles of “mixed” peaks beginning just upstream of the cleavage site and extending downstream to it. These results suggest that the PGC explant has been successfully mutagenized and that the remainder of the embryo (carcass) can be used as a proxy for the PGCs to determine which embryos carrying leapfrog transplants likely have successfully CRISPR/Cas9 mutated 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.
	dazl (deleted in azoospermia-like) gene expression in bissected Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 9, dorsal right.

References:

Ayadi, 2012, Pubmed [+]