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Journal of Assisted Reproduction and Genetics

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01-05-2016 | Technological Innovations

Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing

Authors: Xiangjin Kang, Wenyin He, Yuling Huang, Qian Yu, Yaoyong Chen, Xingcheng Gao, Xiaofang Sun, Yong Fan

Published in: Journal of Assisted Reproduction and Genetics | Issue 5/2016

Abstract

Purpose

As a powerful technology for genome engineering, the CRISPR/Cas system has been successfully applied to modify the genomes of various species. The purpose of this study was to evaluate the technology and establish principles for the introduction of precise genetic modifications in early human embryos.

Methods

3PN zygotes were injected with Cas9 messenger RNA (mRNA) (100 ng/μl) and guide RNA (gRNA) (50 ng/μl). For oligo-injections, donor oligo-1 (99 bp) or oligo-2 (99 bp) (100 ng/μl) or dsDonor (1 kb) was mixed with Cas9 mRNA (100 ng/μl) and gRNA (50 ng/μl) and injected into the embryos.

Results

By co-injecting Cas9 mRNA, gRNAs, and donor DNA, we successfully introduced the naturally occurring CCR5Δ32 allele into early human 3PN embryos. In the embryos containing the engineered CCR5Δ32 allele, however, the other alleles at the same locus could not be fully controlled because they either remained wild type or contained indel mutations.

Conclusions

This work has implications for the development of therapeutic treatments of genetic disorders, and it demonstrates that significant technical issues remain to be addressed. We advocate preventing any application of genome editing on the human germline until after a rigorous and thorough evaluation and discussion are undertaken by the global research and ethics communities.

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Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature. 2001;409(6822):860–921.CrossRefPubMed

Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, et al. The sequence of the human genome. Science. 2001;291(5507):1304–51.CrossRefPubMed

Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. Genome editing with engineered zinc finger nucleases. Nat Rev Genet. 2010;11(9):636–46.CrossRefPubMed

Bogdanove AJ, Voytas DF. TAL effectors: customizable proteins for DNA targeting. Science. 2011;333(6051):1843–6.CrossRefPubMed

Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157(6):1262–78.CrossRefPubMedPubMedCentral

Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910–8.CrossRefPubMedPubMedCentral

Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 2013;154(6):1370–9.CrossRefPubMedPubMedCentral

Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell. 2013;13(6):659–62.CrossRefPubMed

Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science. 2014;345(6201):1184–8.CrossRefPubMedPubMedCentral

10.

Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell. 2014;156(4):836–43.CrossRefPubMed

11.

Liu H, Chen Y, Niu Y, Zhang K, Kang Y, Ge W, et al. TALEN-mediated gene mutagenesis in rhesus and cynomolgus monkeys. Cell Stem Cell. 2014;14:323–8.CrossRefPubMedPubMedCentral

12.

Liang P, Xu Y, Zhang X, Ding C, Huang R, Zhang Z, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015;6(5):363–72.CrossRefPubMedPubMedCentral

13.

Liu P, Chen S, Li X, Qin L, Huang K, Wang L et al. Low immunogenicity of neural progenitor cells differentiated from induced pluripotent stem cells derived from less immunogenic somatic cells. PLoS One. 8(7):e69617.

14.

Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 339(6121):819–23.

15.

Kola I, Trounson A, Dawson G, Rogers P. Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos. Biol Reprod. 1987;37(2):395–401.CrossRefPubMed

16.

Feenan K, Herbert M. Can “abnormally” fertilized zygotes give rise to viable embryos? Hum Fertil. 2006;9(3):157–69.CrossRef

17.

Balakier H. Tripronuclear human zygotes: the first cell cycle and subsequent development. Hum Reprod. 1993;8(11):1892–7.CrossRefPubMed

18.

Martinson JJ, Chapman NH, Rees DC, Liu YT, Clegg JB. Global distribution of the CCR5 gene 32-basepair deletion. Nat Genet. 1997;16(1):100–3.CrossRefPubMed

19.

Marmor M, Sheppard HW, Donnell D, Bozeman S, Celum C, Buchbinder S, et al. Homozygous and heterozygous CCR5-Delta32 genotypes are associated with resistance to HIV infection. J Acquir Immune Defic Syndr. 2001;27(5):472–81.CrossRefPubMed

20.

Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber CM, et al. Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature. 1996;382(6593):722–5.CrossRefPubMed

21.

Holt N, Wang J, Kim K, Friedman G, Wang X, Taupin V, et al. Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat Biotechnol. 2010;28(8):839–47.CrossRefPubMedPubMedCentral

22.

Li L, Krymskaya L, Wang J, Henley J, Rao A, Cao LF, et al. Genomic editing of the HIV-1 coreceptor CCR5 in adult hematopoietic stem and progenitor cells using zinc finger nucleases. Mol Ther : J Am Soc Gene Ther. 2013;21(6):1259–69.CrossRef

23.

Didigu CA, Wilen CB, Wang J, Duong J, Secreto AJ, Danet-Desnoyers GA, et al. Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection. Blood. 2014;123(1):61–9.CrossRefPubMedPubMedCentral

24.

Yao Y, Nashun B, Zhou T, Qin L, Qin L, Zhao S, et al. Generation of CD34+ cells from CCR5-disrupted human embryonic and induced pluripotent stem cells. Hum Gene Ther. 2012;23(2):238–42.CrossRefPubMed

25.

Ye L, Wang J, Beyer AI, Teque F, Cradick TJ, Qi Z, et al. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Delta32 mutation confers resistance to HIV infection. Proc Natl Acad Sci U S A. 2014;111(26):9591–6.CrossRefPubMedPubMedCentral

26.

Ramalingam S, London V, Kandavelou K, Cebotaru L, Guggino W, Civin C, et al. Generation and genetic engineering of human induced pluripotent stem cells using designed zinc finger nucleases. Stem Cells Dev. 2013;22(4):595–610.CrossRefPubMed

27.

Tesson L, Usal C, Menoret S, Leung E, Niles BJ, Remy S, et al. Knockout rats generated by embryo microinjection of TALENs. Nat Biotechnol. 2011;29(8):695–6.CrossRefPubMed

28.

Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31:827–32.CrossRefPubMedPubMedCentral

29.

Hong SG, Winkler T, Wu C, Guo V, Pittaluga S, Nicolae A et al. Path to the clinic: assessment of iPSC-based cell therapies in vivo in a nonhuman primate model. Cell Rep. 7(4):1298–309.

30.

Frock RL, Hu J, Meyers RM, Ho YJ, Kii E, Alt FW. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat Biotechnol. 2015;33(2):179–86.CrossRefPubMed

31.

Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015;33(2):187–97.CrossRefPubMed

32.

Kim D, Bae S, Park J, Kim E, Kim S, Yu HR, et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015;12(3):237–43.CrossRefPubMed

33.

Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154(6):1380–9.CrossRefPubMedPubMedCentral

34.

Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol. 2014;32(6):569–76.CrossRefPubMedPubMedCentral

35.

Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 2014;32(3):279–84.CrossRefPubMedPubMedCentral

36.

Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34(2):184–91.CrossRefPubMedPubMedCentral

37.

Yu C, Liu Y, Ma T, Liu K, Xu S, Zhang Y, et al. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell. 2015;16(2):142–7.CrossRefPubMedPubMedCentral

38.

Wu Y, Zhou H, Fan X, Zhang Y, Zhang M, Wang Y, et al. Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells. Cell Res. 2015;25(1):67–79.CrossRefPubMed

39.

Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, et al. Biotechnology: a prudent path forward for genomic engineering and germline gene modification. Science. 2015;348(6230):36–8.CrossRefPubMedPubMedCentral

Title: Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing
Authors: Xiangjin Kang
Wenyin He
Yuling Huang
Qian Yu
Yaoyong Chen
Xingcheng Gao
Xiaofang Sun
Yong Fan
Publication date: 01-05-2016
Publisher: Springer US
Published in: Journal of Assisted Reproduction and Genetics / Issue 5/2016
Print ISSN: 1058-0468
Electronic ISSN: 1573-7330
DOI: https://doi.org/10.1007/s10815-016-0710-8

ACC 2024 Congress

Springer Medicine

Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing

Abstract

Purpose

Methods

Results

Conclusions

ACC 2024 Congress

Springer Medicine

Abstract

Purpose

Methods

Results

Conclusions

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