Transgenic Research

, Volume 27, Issue 6, pp 525–537 | Cite as

Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep

  • Marcela Vilarino
  • Fabian Patrik Suchy
  • Sheikh Tamir Rashid
  • Helen Lindsay
  • Juan Reyes
  • Bret Roberts McNabb
  • Talitha van der Meulen
  • Mark O. Huising
  • Hiromitsu NakauchiEmail author
  • Pablo Juan RossEmail author
Original Paper


The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in ~ 50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.


Gene-editing Livestock Biopsy Mosaic Ovine 



We would like to thank Alma Islas-Trejo for library preparation and Elizabeth Tseng for assistance in bioinformatics. We also would like to acknowledge Kyle Wood for assistance with sheep care at UC Davis sheep Facility; and Devon Fitzpatrick, Ahmed Mahdi, Michelle Cruz, Charnice Robinson for helping during in vitro embryo production, embryo transferring and fetuses recovering. M.V was supported by a Fulbright-Uruguay Scholarship and an Austin Eugene Lyons Fellowship. Work was partially supported by USDA-NIFA-AFRI multistate project W3171 to P.J.R.

Author’s contribution

MV and FPS performed the experiments with additional input from STR, PJR and HN. MV, FPS, PJR, HL, JR, BRM, TM and MOH participated in sample processing and data analysis. MV, FS and PJR wrote the manuscript with suggestions from all the co-authors.

Compliance with ethical standards

Conflict of interest

Authors declare no competing financial interests statement.

Supplementary material

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Supplementary material 1 (DOCX 19 kb)
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Supplementary material 2 (PDF 835 kb)
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Supplementary material 3 (PDF 151 kb)
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Supplementary material 4 (PDF 157 kb)
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Supplementary material 5 (PDF 157 kb)


  1. Bogliotti YS, Vilarino M, Ross PJ (2016) Laser-assisted cytoplasmic microinjection in livestock zygotes. J Vis Exp. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Burkard C et al (2017) Precision engineering for PRRSV resistance in pigs: macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function. PLoS Pathog 13:e1006206. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Capalbo A, Ubaldi FM, Rienzi L, Scott R, Treff N (2017) Detecting mosaicism in trophectoderm biopsies: current challenges and future possibilities. Hum Reprod 32:492–498. CrossRefPubMedGoogle Scholar
  4. Cimadomo D, Capalbo A, Ubaldi FM, Scarica C, Palagiano A, Canipari R, Rienzi L (2016) The impact of biopsy on human embryo developmental potential during preimplantation genetic diagnosis. Biomed Res Int 2016:7193075. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Dreesen J et al (2008) Validation of preimplantation genetic diagnosis by PCR analysis: genotype comparison of the blastomere and corresponding embryo, implications for clinical practice. Mol Hum Reprod 14:573–579. CrossRefPubMedGoogle Scholar
  6. Gaj T, Gersbach CA, Barbas CF 3rd (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Hai T, Teng F, Guo R, Li W, Zhou Q (2014) One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res 24:372–375. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Hongbing HAN, Tao WANG, Ling LIAN, Xiuzhi TIAN, Rui HU, Shoulong DENG, Kongpan LI, Feng WANG, Ning LI, Guoshi LIU, Yaofeng ZHAO, Zhengxing LIAN (2014) One-step generation of myostatin gene knockout sheep via the CRISPR/Cas9 system. Front Agric Sci Eng 1:2–5. CrossRefGoogle Scholar
  9. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. CrossRefGoogle Scholar
  10. Kobayashi T et al (2010) Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell 142:787–799. CrossRefPubMedGoogle Scholar
  11. Lauri A et al (2013) Assessment of MDA efficiency for genotyping using cloned embryo biopsies. Genomics 101:24–29. CrossRefPubMedGoogle Scholar
  12. Li H (2018) Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Lindsay H et al (2016) CrispRVariants charts the mutation spectrum of genome engineering experiments. Nat Biotechnol 34:701–702. CrossRefPubMedGoogle Scholar
  14. Midic U et al (2017) Quantitative assessment of timing, efficiency, specificity and genetic mosaicism of CRISPR/Cas9-mediated gene editing of hemoglobin beta gene in rhesus monkey embryos. Hum Mol Genet 26:2678–2689. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Niu Y et al (2017) Biallelic beta-carotene oxygenase 2 knockout results in yellow fat in sheep via CRISPR/Cas9. Anim Genet 48:242–244. CrossRefPubMedGoogle Scholar
  16. Piyamongkol W, Bermudez MG, Harper JC, Wells D (2003) Detailed investigation of factors influencing amplification efficiency and allele drop-out in single cell PCR: implications for preimplantation genetic diagnosis. Mol Hum Reprod 9:411–420CrossRefGoogle Scholar
  17. Sato M et al (2018) Timing of CRISPR/Cas9-related mRNA microinjection after activation as an important factor affecting genome editing efficiency in porcine oocytes. Theriogenology 108:29–38. CrossRefPubMedGoogle Scholar
  18. Suchy F, Nakauchi H (2017) Lessons from Interspecies Mammalian Chimeras. Annu Rev Cell Dev Biol 33:203–217. CrossRefPubMedGoogle Scholar
  19. Suchy F, Yamaguchi T, Nakauchi H (2018) iPSC-derived organs in vivo: challenges and promise. Cell Stem Cell 22:21–24. CrossRefPubMedGoogle Scholar
  20. Tan W, Proudfoot C, Lillico SG, Whitelaw CB (2016) Gene targeting, genome editing: from Dolly to editors. Transgenic Res 25:273–287. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Tu Z et al (2017) Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos. Sci Rep 7:42081. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Usui J, Kobayashi T, Yamaguchi T, Knisely AS, Nishinakamura R, Nakauchi H (2012) Generation of kidney from pluripotent stem cells via blastocyst complementation. Am J Pathol 180:2417–2426. CrossRefPubMedGoogle Scholar
  23. Vilarino M et al (2017) CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep. Sci Rep 7:17472. CrossRefPubMedPubMedCentralGoogle Scholar
  24. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Wang X et al (2015) Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs. Sci Rep 5:13348. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Wang K et al (2017) CRISPR/Cas9-mediated knockout of myostatin in Chinese indigenous Erhualian pigs. Transgenic Res 26:799–805. CrossRefPubMedGoogle Scholar
  27. Wu J, Greely HT, Jaenisch R, Nakauchi H, Rossant J, Belmonte JC (2016) Stem cells and interspecies chimaeras. Nature 540:51–59. CrossRefPubMedGoogle Scholar
  28. Wu J et al (2017a) Interspecies chimerism with mammalian pluripotent stem cells. Cell 168(473–486):e415. CrossRefGoogle Scholar
  29. Wu J et al (2017b) CRISPR-Cas9 mediated one-step disabling of pancreatogenesis in pigs. Sci Rep 7:10487. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Yamaguchi T et al (2017) Interspecies organogenesis generates autologous functional islets. Nature 542:191–196. CrossRefPubMedGoogle Scholar
  31. Yen ST et al (2014) Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev Biol 393:3–9. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Zhou J et al (2014) One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering. Int J Biochem Cell Biol 46:49–55. CrossRefPubMedGoogle Scholar
  33. Zuo E et al (2017) One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs. Cell Res 27:933–945. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Department of Animal Science, College of Agricultural and Environmental SciencesUniversity of California DavisDavisUSA
  2. 2.School of Medicine, Institute for Stem Cell Biology and Regenerative MedicineStanford UniversityStanfordUSA
  3. 3.Centre for Stem Cells and Regenerative Medicine and Institute for Liver StudiesKing’s CollegeLondonUK
  4. 4.Institute of Molecular Life SciencesUniversity of ZürichZurichSwitzerland
  5. 5.SIB Swiss Institute of BioinformaticsUniversity of ZürichZurichSwitzerland
  6. 6.Department of Population Health and Reproduction, School of Veterinary MedicineUniversity of California DavisDavisUSA
  7. 7.Department of Neurobiology, Physiology and Behavior, College of Biological SciencesUniversity of California DavisDavisUSA

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