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Development. 2016 Aug 1;143(15):2868-75. doi: 10.1242/dev.138057. Epub 2016 Jul 6.

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

Author information

1
4410 Natural Sciences Building 2, Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA ilblitz@uci.edu.
2
4410 Natural Sciences Building 2, Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA.

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

PMID:
27385011
PMCID:
PMC5004912
DOI:
10.1242/dev.138057
[Indexed for MEDLINE]
Free PMC Article

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