Abstract
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.
Similar content being viewed by others
References
Bogliotti YS, Vilarino M, Ross PJ (2016) Laser-assisted cytoplasmic microinjection in livestock zygotes. J Vis Exp. https://doi.org/10.3791/54465
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. https://doi.org/10.1371/journal.ppat.1006206
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. https://doi.org/10.1093/humrep/dew250
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. https://doi.org/10.1155/2016/7193075
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. https://doi.org/10.1093/molehr/gan052
Gaj T, Gersbach CA, Barbas CF 3rd (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405. https://doi.org/10.1016/j.tibtech.2013.04.004
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. https://doi.org/10.1038/cr.2014.11
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. https://doi.org/10.15302/j-fase-2014007
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. https://doi.org/10.1126/science.1225829
Kobayashi T et al (2010) Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell 142:787–799. https://doi.org/10.1016/j.cell.2010.07.039
Lauri A et al (2013) Assessment of MDA efficiency for genotyping using cloned embryo biopsies. Genomics 101:24–29. https://doi.org/10.1016/j.ygeno.2012.09.002
Li H (2018) Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. https://doi.org/10.1093/bioinformatics/bty191
Lindsay H et al (2016) CrispRVariants charts the mutation spectrum of genome engineering experiments. Nat Biotechnol 34:701–702. https://doi.org/10.1038/nbt.3628
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. https://doi.org/10.1093/hmg/ddx154
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. https://doi.org/10.1111/age.12515
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 9:411–420
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. https://doi.org/10.1016/j.theriogenology.2017.11.030
Suchy F, Nakauchi H (2017) Lessons from Interspecies Mammalian Chimeras. Annu Rev Cell Dev Biol 33:203–217. https://doi.org/10.1146/annurev-cellbio-100616-060654
Suchy F, Yamaguchi T, Nakauchi H (2018) iPSC-derived organs in vivo: challenges and promise. Cell Stem Cell 22:21–24. https://doi.org/10.1016/j.stem.2017.12.003
Tan W, Proudfoot C, Lillico SG, Whitelaw CB (2016) Gene targeting, genome editing: from Dolly to editors. Transgenic Res 25:273–287. https://doi.org/10.1007/s11248-016-9932-x
Tu Z et al (2017) Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos. Sci Rep 7:42081. https://doi.org/10.1038/srep42081
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. https://doi.org/10.1016/j.ajpath.2012.03.007
Vilarino M et al (2017) CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep. Sci Rep 7:17472. https://doi.org/10.1038/s41598-017-17805-0
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. https://doi.org/10.1016/j.cell.2013.04.025
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. https://doi.org/10.1038/srep13348
Wang K et al (2017) CRISPR/Cas9-mediated knockout of myostatin in Chinese indigenous Erhualian pigs. Transgenic Res 26:799–805. https://doi.org/10.1007/s11248-017-0044-z
Wu J, Greely HT, Jaenisch R, Nakauchi H, Rossant J, Belmonte JC (2016) Stem cells and interspecies chimaeras. Nature 540:51–59. https://doi.org/10.1038/nature20573
Wu J et al (2017a) Interspecies chimerism with mammalian pluripotent stem cells. Cell 168(473–486):e415. https://doi.org/10.1016/j.cell.2016.12.036
Wu J et al (2017b) CRISPR-Cas9 mediated one-step disabling of pancreatogenesis in pigs. Sci Rep 7:10487. https://doi.org/10.1038/s41598-017-08596-5
Yamaguchi T et al (2017) Interspecies organogenesis generates autologous functional islets. Nature 542:191–196. https://doi.org/10.1038/nature21070
Yen ST et al (2014) Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev Biol 393:3–9. https://doi.org/10.1016/j.ydbio.2014.06.017
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. https://doi.org/10.1016/j.biocel.2013.10.010
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. https://doi.org/10.1038/cr.2017.81
Acknowledgements
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 information
Authors and Affiliations
Contributions
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.
Corresponding authors
Ethics declarations
Conflict of interest
Authors declare no competing financial interests statement.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Vilarino, M., Suchy, F.P., Rashid, S.T. et al. Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep. Transgenic Res 27, 525–537 (2018). https://doi.org/10.1007/s11248-018-0094-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-018-0094-x