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Super-Mendelian inheritance mediated by CRISPR–Cas9 in the female mouse germline

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An Author Correction to this article was published on 08 January 2020

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Abstract

A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR–Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity1,2,3,4. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR–Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR–Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.

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Fig. 1: Embryonic Cas9 activity does not copy the TyrCopyCat allele from the donor to the receiver chromosome.
Fig. 2: Breeding strategy to produce TyrCopyCat/ch mice with a conditional cas9 transgene and a germline restricted cre transgene.
Fig. 3: Gene conversion by an active genetic element was observed in the female germline and not in the male germline or in the early embryo.

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Data availability

All genotyping data for F3 offspring of constitutive crosses and F4 offspring of germline conditional crosses is available at Zenodo with the identifier https://doi.org/10.5281/zenodo.2003087. Annotated sequence data for the TyrCopyCat transgene is available in GenBank with the accession number MK160997.

Change history

  • 08 January 2020

    An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

References

  1. Gantz, V. M. & Bier, E. The mutagenic chain reaction: a method for converting heterozygous to homozygous mutations. Science 348, 442–444 (2015).

    Article  ADS  CAS  Google Scholar 

  2. Gantz, V. M. et al. Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi. Proc. Natl Acad. Sci. USA 112, E6736–E6743 (2015).

    Article  ADS  CAS  Google Scholar 

  3. Hammond, A. et al. A CRISPR–Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat. Biotechnol. 34, 78–83 (2016).

    Article  CAS  Google Scholar 

  4. Kyrou, K. et al. A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes. Nat. Biotechnol. 36, 1062–1066 (2018).

    Article  CAS  Google Scholar 

  5. Gantz, V. M. & Bier, E. The dawn of active genetics. BioEssays 38, 50–63 (2016).

    Article  Google Scholar 

  6. Gould, F. Broadening the application of evolutionarily based genetic pest management. Evolution 62, 500–510 (2008).

    Article  Google Scholar 

  7. Esvelt, K. M., Smidler, A. L., Catteruccia, F. & Church, G. M. Emerging technology: concerning RNA-guided gene drives for the alteration of wild populations. eLife 3, e03401 (2014).

    Article  Google Scholar 

  8. Mao, Z., Bozzella, M., Seluanov, A. & Gorbunova, V. Comparison of nonhomologous end joining and homologous recombination in human cells. DNA Repair (Amst.) 7, 1765–1771 (2008).

    Article  CAS  Google Scholar 

  9. Miyaoka, Y. et al. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci. Rep. 6, 23549 (2016).

    Article  ADS  CAS  Google Scholar 

  10. Xu, X.-R. S., Gantz, V. M., Siomava, N. & Bier, E. CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity. eLife 6, e30281 (2017).

    Article  Google Scholar 

  11. Yokoyama, T. et al. Conserved cysteine to serine mutation in tyrosinase is responsible for the classical albino mutation in laboratory mice. Nucleic Acids Res. 18, 7293–7298 (1990).

    Article  CAS  Google Scholar 

  12. Yen, S.-T. et al. Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev. Biol. 393, 3–9 (2014).

    Article  CAS  Google Scholar 

  13. Miyagishi, M. & Taira, K. U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat. Biotechnol. 20, 497–500 (2002).

    Article  CAS  Google Scholar 

  14. Boshart, M. et al. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 41, 521–530 (1985).

    Article  CAS  Google Scholar 

  15. Platt, R. J. et al. CRISPR–Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014).

    Article  CAS  Google Scholar 

  16. Chiou, S.-H. et al. Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing. Genes Dev. 29, 1576–1585 (2015).

    Article  CAS  Google Scholar 

  17. Beermann, F. et al. Rescue of the albino phenotype by introduction of a functional tyrosinase gene into mice. EMBO J. 9, 2819–2826 (1990).

    Article  CAS  Google Scholar 

  18. Keeney, S., Giroux, C. N. & Kleckner, N. Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88, 375–384 (1997).

    Article  CAS  Google Scholar 

  19. Goedecke, W., Eijpe, M., Offenberg, H. H., van Aalderen, M. & Heyting, C. Mre11 and Ku70 interact in somatic cells, but are differentially expressed in early meiosis. Nat. Genet. 23, 194–198 (1999).

    Article  CAS  Google Scholar 

  20. Gallardo, T., Shirley, L., John, G. B. & Castrillon, D. H. Generation of a germ cell-specific mouse transgenic Cre line, Vasa-cre. Genesis 45, 413–417 (2007).

    Article  CAS  Google Scholar 

  21. Sadate-Ngatchou, P. I., Payne, C. J., Dearth, A. T. & Braun, R. E. Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice. Genesis 46, 738–742 (2008).

    Article  CAS  Google Scholar 

  22. de Rooij, D. G. & Grootegoed, J. A. Spermatogonial stem cells. Curr. Opin. Cell Biol. 10, 694–701 (1998).

    Article  Google Scholar 

  23. Pepling, M. E. From primordial germ cell to primordial follicle: mammalian female germ cell development. Genesis 44, 622–632 (2006).

    Article  CAS  Google Scholar 

  24. Burt, A. Site-specific selfish genes as tools for the control and genetic engineering of natural populations. Proc. R. Soc. Lond. B 270, 921–928 (2003).

    Article  CAS  Google Scholar 

  25. Deredec, A., Burt, A. & Godfray, H. C. J. The population genetics of using homing endonuclease genes in vector and pest management. Genetics 179, 2013–2026 (2008).

    Article  Google Scholar 

  26. Unckless, R. L., Clark, A. G. & Messer, P. W. Evolution of resistance against CRISPR/Cas9 gene drive. Genetics 205, 827–841 (2017).

    Article  Google Scholar 

  27. Noble, C., Olejarz, J., Esvelt, K. M., Church, G. M. & Nowak, M. A. Evolutionary dynamics of CRISPR gene drives. Sci. Adv. 3, e1601964 (2017).

    Article  ADS  Google Scholar 

  28. Marshall, J. M., Buchman, A., Sánchez C, H. M. & Akbari, O. S. Overcoming evolved resistance to population-suppressing homing-based gene drives. Sci. Rep. 7, 3776 (2017).

    Article  ADS  Google Scholar 

  29. Noble, C., Adlam, B., Church, G. M., Esvelt, K. M. & Nowak, M. A. Current CRISPR gene drive systems are likely to be highly invasive in wild populations. eLife 7, e33423 (2018).

    Article  Google Scholar 

  30. Jensen-Seaman, M. I. et al. Comparative recombination rates in the rat, mouse, and human genomes. Genome Res. 14, 528–538 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K. Hanley for the DNA extraction protocol; A. Green and A.-C. Chen for genotyping assistance; M. Tran for laser-capture microdissection in an effort to genotype spermatogonia; P. Jain for assistance with fibroblast transfection; H. Cook-Andersen and M. Wilkinson for conversations about mouse germline development; L. Montoliu for discussion of the tyrosinase locus; M. Tuszynski for plasmids and for early support of the project. This work was funded by a Searle Scholar Award from the Kinship Foundation, a Pew Biomedical Scholar Award from the Pew Charitable Trusts, a Packard Fellowship in Science and Engineering from the David and Lucile Packard Foundation, and NIH grant R21GM129448 awarded to K.L.C. E.B. was supported by NIH grant R01GM117321, a Paul G. Allen Frontiers Group Distinguished Investigators Award and a gift from the Tata Trusts in India to TIGS-UCSD and TIGS-India. H.A.G. was supported by a Ruth Stern Graduate Fellowship and by the NIH Cell and Molecular Genetics training grant T32GM724039; V.M.G. was supported by NIH grant DP5OD023098.

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Nature thanks B. Conklin, S. Qi and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Author notes

  1. Gunnar Poplawski

    Present address: Department of Medicine, National University of Singapore, Singapore, Singapore

  2. These authors contributed equally: Hannah A. Grunwald, Valentino M. Gantz, Gunnar Poplawski

Authors and Affiliations

  1. Division of Biological Sciences, Section of Cellular and Developmental Biology, University of California, San Diego, La Jolla, CA, USA

    Hannah A. Grunwald, Valentino M. Gantz, Xiang-Ru S. Xu, Ethan Bier & Kimberly L. Cooper

  2. Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA

    Gunnar Poplawski

  3. Tata Institute for Genetics and Society, University of California, San Diego, La Jolla, CA, USA

    Ethan Bier & Kimberly L. Cooper

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  1. Hannah A. Grunwald
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Contributions

H.A.G., V.M.G., G.P., E.B. and K.L.C. conceived and designed the research; V.M.G. and G.P. designed and cloned the TyrCopyCat transgene and validated mCherry expression in vitro; X.-R.S.X. validated Tyr4a gRNA cleavage activity in vitro; H.A.G. and K.L.C. designed the breeding strategies; H.A.G. acquired and established mouse lines, developed genotyping protocols, and conducted mouse breeding, phenotyping and genotyping; H.A.G. curated the data and analyses; K.L.C. and E.B. supervised the research; K.L.C. wrote the original draft; H.A.G., V.M.G., E.B. and K.L.C. revised subsequent drafts.

Corresponding author

Correspondence to Kimberly L. Cooper.

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Competing interests

V.M.G., E.B. and K.L.C. hold advisory board positions with Synbal. All other authors declare that they have no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Knock-in strategy using the TyrCopyCat-targeting vector.

The U6-Tyr4a gRNA (tyrosinase exon 4 gRNA a) and CMV-mCherry were inserted into the cut site of the Tyr4a gRNA by HDR after CRISPR–Cas9 DSB formation targeted by the Tyr4a gRNA. See Supplementary Methods and Supplementary Figs. 1, 2 for additional details.

Extended Data Fig. 2 Rosa26-cas9 and H11-cas9 constitutive lineages have different numbers of unique NHEJ indels.

Sanger sequencing of the Tyr4a gRNA target exon amplified from tail-tip genomic DNA using TyrHALF2 and TyrHARR2 primers as specified in Supplementary Table 3. Top, a single representative Sanger sequence trace of the bulk PCR product amplified from a Rosa26-cas9;TyrCopyCat-positive F2 mouse (Rosa26 family 1 in Extended Data Table 3) with either major or minor peaks called revealing two distinct alleles. Five Tyrch-positive F3 offspring of this F2 individual each match one of the two alleles (marked 1 (insertion) and 2 (deletion)). Bottom, a single representative sequence trace of the bulk PCR product amplified from an H11-cas9;TyrCopyCat-positive F2 mouse (H11 family 1 in Extended Data Table 3). Alternate alleles cannot be called because of the complexity of overlapping peaks. Five Tyrch-positive F3 offspring each have one of four different alleles (marked 1, 2, 3 and 4). Sequence trace data are representative of all 90 individuals of 5 families of each constitutive strategy described in Extended Data Table 3.

Extended Data Table 1 Coat colour of F2 individuals that were constitutive cas9-positive and TyrCopyCat/ch
Full size table
Extended Data Table 2 Analyses of phenotypes and genotypes of all F3 progeny of the constitutive cas9 crosses
Full size table
Extended Data Table 3 Allelic complexity of the constitutive Rosa26- and H11-cas9 families
Full size table
Extended Data Table 4 Analyses of phenotypes and genotypes of all F4 progeny of the germline Cas9 crosses
Full size table

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Figures 1-5 and Supplementary Tables 1-4. Supplementary Methods detail the TyrCopyCat targeted insertion into the mouse genome and genotyping methods for all alleles together with supporting Supplementary Figures. Supplementary Tables include all primers and mouse lines used in this study and information about the distribution of coat colors among F3 parents of each germline strategy.

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Grunwald, H.A., Gantz, V.M., Poplawski, G. et al. Super-Mendelian inheritance mediated by CRISPR–Cas9 in the female mouse germline. Nature 566, 105–109 (2019). https://doi.org/10.1038/s41586-019-0875-2

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