Asymmetric parental genome engineering by Cas9 during mouse meiotic exit.

Toru Suzuki,Anthony C F Perry,Maki Asami

doi:10.1038/srep07621

Toru Suzuki, Anthony C F Perry + Show 1 more

Open Access

https://doi.org/10.1038/srep07621

Copy DOI

Journal: Scientific reports	Publication Date: Dec 17, 2014
Citations: 50	License type: CC BY 4.0

Affiliation: University of Bath

Abstract

Mammalian genomes can be edited by injecting pronuclear embryos with Cas9 cRNA and guide RNA (gRNA) but it is unknown whether editing can also occur during the onset of embryonic development, prior to pronuclear embryogenesis. We here report Cas9-mediated editing during sperm-induced meiotic exit and the initiation of development. Injection of unfertilized, mouse metaphase II (mII) oocytes with Cas9 cRNA, gRNA and sperm enabled efficient editing of transgenic and native alleles. Pre-loading oocytes with Cas9 increased sensitivity to gRNA ~100-fold. Paternal allelic editing occurred as an early event: single embryo genome analysis revealed editing within 3 h of sperm injection, coinciding with sperm chromatin decondensation during the gamete-to-embryo transition but prior to pronucleus formation. Maternal alleles underwent editing after the first round of DNA replication, resulting in mosaicism. Asymmetric editing of maternal and paternal alleles suggests a novel strategy for discriminatory targeting of parental genomes.

Highlights

Laboratory of Mammalian Molecular Embryology, Department of Biology and Biochemistry, University of Bath, BA2 7AY, England
Paternal allelic editing occurred as an early event: single embryo genome analysis revealed editing within 3 h of sperm injection, coinciding with sperm chromatin decondensation during the gamete-to-embryo transition but prior to pronucleus formation
Sperm combine with metaphase II oocytes and become denuded of nucleoprotein in a process termed decondensation, before their genomes recondense with maternal histones www.nature.com/scientificreports and form a nuclear structure, the pronucleus15–17. (The maternal genome simultaneously forms a separate pronucleus.) The early phase in which sperm decondensation occurs coincides with the gamete-to-embryo transition and supports efficient transgene integration at quasi-random sites[18,19]; transgenes preferentially integrate into the sperm-derived genome, possibly because protein depletion during sperm decondensation makes it a better substrate for recombination[17]

Summary

Results and discussion

To evaluate genome editing during meiotic exit, we first generated a test-bed transgenic line (129-eGFP) by introducing a single-copy ubiquitously-expressed pCAG-eGFP transgene (eGFP) onto the 129/Sv background. These results were confirmed by analogous experiments with a Nanog-eGFP knock-in line (Fig. 1E and Supplementary Fig. S1A) This shows that genome targeting by injecting mII oocytes with sperm, Cas[9] cRNA and gRNA can be achieved high efficiencies and is not locus-specific. Mutations were transmitted to offspring, including inheritance of the non-pigmented eye phenotype in F1 progeny of coat colour founders (with overtly normal eyes), indicating that coat colour and eye phenotypes have a shared aetiology and further revealing founder mosaicism (Supplementary Fig. S2 and Supplementary Fig. S3B) These experiments demonstrate that injecting mII oocytes with Cas[9] cRNA, gRNA and sperm efficiently produces embryos and offspring with edited genomes. This formal possibility will require exhaustive evaluation, but if successful, could enable genomic surgery for gene repair during the initiation of embryogenesis

Methods

Author contributions

Additional information