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CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein

Abstract

Previous works using human tripronuclear zygotes suggested that the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system could be a tool in correcting disease-causing mutations. However, whether this system was applicable in normal human (dual pronuclear, 2PN) zygotes was unclear. Here we demonstrate that CRISPR/Cas9 is also effective as a gene-editing tool in human 2PN zygotes. By injection of Cas9 protein complexed with the appropriate sgRNAs and homology donors into one-cell human embryos, we demonstrated efficient homologous recombination-mediated correction of point mutations in HBB and G6PD. However, our results also reveal limitations of this correction procedure and highlight the need for further research.

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References

  1. Capmany G, Taylor A, Braude PR, Bolton VN (1996) The timing of pronuclear formation, DNA synthesis and cleavage in the human 1-cell embryo. Mol Hum Reprod 2:299–306

  2. Chapman JR, Taylor MR, Boulton SJ (2012) Playing the end game: DNA double-strand break repair pathway choice. Mol Cell 47:497–510

  3. Chen F, Pruett-Miller SM, Huang Y, Gjoka M, Duda K, Taunton J, Collingwood TN, Frodin M, Davis GD (2011) High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat Methods 8:753–755

  4. Chen S, Lee B, Lee AY, Modzelewski AJ, He L (2016) Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J Biol Chem 291:14457–14467

  5. Cho SW, Lee J, Carroll D, Kim JS, Lee J (2013) Heritable gene knockout in Caenorhabditis elegans by direct injection of Cas9-sgRNA ribonucleoproteins. Genetics 195:1177–1180

  6. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823

  7. Du CS, Ren X, Chen L, Jiang W, He Y, Yang M (1999) Detection of the most common G6PD gene mutations in Chinese using amplification refractory mutation system. Hum Hered 49:133–138

  8. Hashimoto M, Yamashita Y, Takemoto T (2016) Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Dev Biol 418:1–9

  9. Hasty P, Rivera-Perez J, Bradley A (1991) The length of homology required for gene targeting in embryonic stem cells. Mol Cell Biol 11:5586–5591

  10. Heyer WD, Ehmsen KT, Liu J (2010) Regulation of homologous recombination in eukaryotes. Annu Rev Genet 44:113–139

  11. Kang X, He W, Huang Y, Yu Q, Chen Y, Gao X, Sun X, Fan Y (2016) Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing. J Assist Reprod Genet 33:581–588

  12. Kim S, Kim D, Cho SW, Kim J, Kim JS (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24:1012–1019

  13. Kimura A, Matsunaga E, Takihara Y, Nakamura T, Takagi Y, Lin S, Lee H (1983) Structural analysis of a beta-thalassemia gene found in Taiwan. J Biol Chem 258:2748–2749

  14. Lee JS, Kwak SJ, Kim J, Kim AK, Noh HM, Kim JS, Yu K (2014) RNA-guided genome editing in Drosophila with the purified Cas9 protein. G3 (Bethesda) 4:1291–1295

  15. Li W, Teng F, Li T, Zhou Q (2013) Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol 31:684–686

  16. Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, Lv J, Xie X, Chen Y, Li Y, Sun Y, Bai Y, Songyang Z, Ma W, Zhou C, Huang J (2015) CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell 6:363–372

  17. Lieber MR (2010) The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 79:181–211

  18. Lin S, Staahl BT, Alla RK, Doudna JA (2014a) Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife 3:e04766

  19. Lin Y, Cradick TJ, Brown MT, Deshmukh H, Ranjan P, Sarode N, Wile BM, Vertino PM, Stewart FJ, Bao G (2014b) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res 42:7473–7485

  20. Ma Y, Shen B, Zhang X, Lu Y, Chen W, Ma J, Huang X, Zhang L (2014) Heritable multiplex genetic engineering in rats using CRISPR/Cas9. PLoS One 9:e89413

  21. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826

  22. Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J (2014) Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156:836–843

  23. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154:1380–1389

  24. Shen B, Zhang J, Wu H, Wang J, Ma K, Li Z, Zhang X, Zhang P, Huang X (2013) Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res 23:720–723

  25. 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

  26. Zhou J, Shen B, Zhang W, Wang J, Yang J, Chen L, Zhang N, Zhu K, Xu J, Hu B, Leng Q, Huang X (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

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Acknowledgements

We thank Dr. Yuanfeng Li of Beijing Proteome Research Center and Dr. Zhongsheng Sun of Beijing Institutes of Life Science, Chinese Academy of Sciences for help with genome sequencing and analysis. We thank Dr. Xingxu Huang of the School of Life Science and Technology, Shanghai Tech University for helpful advice.

Author information

Correspondence to Lichun Tang or Xiaowei Li or Jianqiao Liu.

Ethics declarations

Funding

This study was funded by a Grant from the Beijing Municipal Committee on Science and Technology (#Z141100000214015), by an international collaboration Grant from the Chinese Minister of Science and Technology (#2013DFB30210), by The National Basic Research Program (973 Program, No. 2013CB910300), by a Grant from the Science and Technology Program of Guangzhou, China (#2014KZDXM047), by The Innovation of Science and Technology Commission of Guangzhou, China (No. 201604020075), by The Department of Science and Technology of Guangdong, China (No. 2016A020218012), and a Grant from the China Postdoctoral Science Foundation (#2013M532214).

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical approval

This study was approved by the ethics committee of the Third Affiliated Hospital of Guangzhou Medical University, numbered [2015] No. 068. The methods used in the present study closely followed the guidelines legislated and posted by the Ministry of Health of the People’s Republic of China. The patients involved in this study knew about and understood the usage of tripronuclear zygotes, immature oocytes and leftover sperm, and voluntarily donated them after providing informed consent.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Additional information

Communicated by S. Hohmann.

Electronic supplementary material

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Supplementary material 1 (DOCX 222 KB)

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Cite this article

Tang, L., Zeng, Y., Du, H. et al. CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein. Mol Genet Genomics 292, 525–533 (2017). https://doi.org/10.1007/s00438-017-1299-z

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Keywords

  • CRISPR/Cas9
  • Homology-directed repair (HDR)
  • Cas9 protein
  • Gene modification
  • Human zygotes