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Cellular and Molecular Life Sciences

, Volume 73, Issue 13, pp 2543–2563 | Cite as

Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system

  • Yan Zhou
  • Yong Liu
  • Dianna Hussmann
  • Peter Brøgger
  • Rasha Abdelkadhem Al-Saaidi
  • Shuang Tan
  • Lin Lin
  • Trine Skov Petersen
  • Guang Qian Zhou
  • Peter Bross
  • Lars Aagaard
  • Tino Klein
  • Sif Groth Rønn
  • Henrik Duelund Pedersen
  • Lars Bolund
  • Anders Lade Nielsen
  • Charlotte Brandt Sørensen
  • Yonglun Luo
Original Article

Abstract

Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check. We opted for the Golden Gate Cloning strategy to simplify C-Check construction. To demonstrate the utility of the C-Check system, we used the C-Check in combination with TALENs or CRISPR/Cas9 in different scenarios of gene editing experiments. First, we disrupted the endogenous pIAPP gene (3.0 % efficiency) by C-Check-validated TALENs in primary porcine fibroblasts (PPFs). Next, we achieved gene-editing efficiencies of 9.0–20.3 and 4.9 % when performing single- and double-gene targeting (MAPT and SORL1), respectively, in PPFs using C-Check-validated CRISPR/Cas9 vectors. Third, fluorescent tagging of endogenous genes (MYH6 and COL2A1, up to 10.0 % frequency) was achieved in human fibroblasts with C-Check-validated CRISPR/Cas9 vectors. We further demonstrated that the C-Check system could be applied to enrich for IGF1R null HEK293T cells and CBX5 null MCF-7 cells with frequencies of nearly 100.0 and 86.9 %, respectively. Most importantly, we further showed that the C-Check system is compatible with multiplexing and for studying CRISPR/Cas9 sgRNA specificity. The C-Check system may serve as an alternative dual-fluorescent surrogate tool for measuring DNA nuclease activity and enrichment of gene-edited cells, and may thereby aid in streamlining programmable DNA nuclease-mediated genome editing and biological research.

Keywords

Dual-fluorescent surrogate reporter TALENs CRISPR/Cas9 Gene targeting Genome engineering Single-strand annealing Homologous recombination 

Notes

Acknowledgments

We are grateful to the FACS CORE facility (with special thanks to Charlotte Christie Petersen) at the Department of Biomedicine, Aarhus University for assistance with flow cytometry and FACS. This work was supported in part by grants from the STAR programme from the R&D Department, Novo Nordisk A/S to YL; and the Danish Research Council for Independent Research (16942) to YL; the Sapere Aude Young Research Talent prize to YL (18382); the Lundbeck Foundation (R173-2014-1105, R151-2013-14439, R126-2012-12448, R100-A9209, R173-2014-993, and R100-A9606) to YL, LB, PB, CBS, and ALN respectively; the China Scholarship Council (CSC) to YZ; the Natural Science Foundation of China (81472126) to ST and GQZ; the Toyota Foundation ALN; and the AUFF AU IDEAS Programme and The Karen Elise Jensen Foundation to CBS.

Compliance with ethical standards

Conflict of interest

YL (2012–2014), SGR, and HD were financed by Novo Nordisk A/S. TK was financed by Gubra ApS. A patent claim is declared to the generation of a diabetes pig model based on genetic modification of the porcine IAPP gene. No other competing interests are declared by the authors.

Supplementary material

18_2015_2128_MOESM1_ESM.txt (13 kb)
Additional File 1: Genebank file of the C-Check vector (TXT 12 kb)
18_2015_2128_MOESM2_ESM.xlsx (44 kb)
Additional File 2: List of TALEN and CRISPR/Cas9 target sites (XLSX 43 kb)
18_2015_2128_MOESM3_ESM.pdf (609 kb)
Additional File 3: Optimization of nucleofection in porcine fibroblasts. Five nucleofection reagents (P1-P5) and 15 nucleofection programs were evaluated (PDF 608 kb)
18_2015_2128_MOESM4_ESM.docx (166 kb)
Additional File 4: List of oligonucleotides and primers (DOCX 166 kb)
18_2015_2128_MOESM5_ESM.pdf (14 mb)
Additional File 5: Sanger sequencing analysis of one IGF1R potential off-target site (PDF 14353 kb)
18_2015_2128_MOESM6_ESM.docx (21 kb)
Additional File 6: Sanger sequencing of IGF1R knockout clonogenic cell clones (DOCX 20 kb)
18_2015_2128_MOESM7_ESM.docx (132 kb)
Additional File 7: Sanger sequencing of CBX5 knockout clonogenic cell clones (DOCX 131 kb)
18_2015_2128_MOESM8_ESM.xlsx (55 kb)
Additional File 8: C-Check CRISPR OFF sgRNA design and sequences (XLSX 54 kb)
18_2015_2128_MOESM9_ESM.docx (111 kb)
Additional File 9: Detailed protocol for construction of the C-Check reporter vector (DOCX 110 kb)
18_2015_2128_MOESM10_ESM.xlsx (42 kb)
Additional File 10: C-Check complementary oligonucleotide design (XLSX 41 kb)

References

  1. 1.
    Gaj T, Guo J, Kato Y, Sirk SJ, Barbas CF 3rd (2012) Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat Methods 9:805–807CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Kim YG, Chandrasegaran S (1994) Chimeric restriction endonuclease. Proc Natl Acad Sci USA 91:883–887CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver RC, Katibah GE, Amora R, Boydston EA, Zeitler B et al (2009) Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 27:851–857CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Hockemeyer D, Wang H, Kiani S, Lai CS, Gao Q, Cassady JP, Cost GJ, Zhang L, Santiago Y, Miller JC et al (2011) Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 29:731–734CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Bogdanove AJ, Voytas DF (2011) TAL effectors: customizable proteins for DNA targeting. Science 333:1843–1846CrossRefPubMedGoogle Scholar
  6. 6.
    Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326:1509–1512CrossRefPubMedGoogle Scholar
  7. 7.
    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 339:823–826CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    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 339:819–823CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Haurwitz RE, Jinek M, Wiedenheft B, Zhou K, Doudna JA (2010) Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329:1355–1358CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ et al (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148CrossRefPubMedGoogle Scholar
  11. 11.
    Bedell VM, Wang Y, Campbell JM, Poshusta TL, Starker CG, Krug RG 2nd, Tan W, Penheiter SG, Ma AC, Leung AY et al (2012) In vivo genome editing using a high-efficiency TALEN system. Nature 491:114–118CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Tesson L, Usal C, Menoret S, Leung E, Niles BJ, Remy S, Santiago Y, Vincent AI, Meng X, Zhang L et al (2011) Knockout rats generated by embryo microinjection of TALENs. Nat Biotechnol 29:695–696CrossRefPubMedGoogle Scholar
  13. 13.
    Carlson DF, Tan W, Lillico SG, Stverakova D, Proudfoot C, Christian M, Voytas DF, Long CR, Whitelaw CB, Fahrenkrug SC (2012) Efficient TALEN-mediated gene knockout in livestock. Proc Natl Acad Sci USA 109:17382–17387CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Li T, Liu B, Spalding MH, Weeks DP, Yang B (2012) High-efficiency TALEN-based gene editing produces disease-resistant rice. Nat Biotechnol 30:390–392CrossRefPubMedGoogle Scholar
  15. 15.
    Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK (2012) FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 30:460–465CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J (2013) RNA-programmed genome editing in human cells. eLife 2:e00471CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    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 337:816–821CrossRefPubMedGoogle Scholar
  18. 18.
    Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang DL, Wang Z, Zhang Z, Zheng R, Yang L et al (2014) Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA 111:4632–4637CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Mandal PK, Ferreira LM, Collins R, Meissner TB, Boutwell CL, Friesen M, Vrbanac V, Garrison BS, Stortchevoi A, Bryder D et al (2014) Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell 15:643–652CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas O, Eisenhaure TM, Jovanovic M et al (2014) CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159:440–455CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Shao Y, Guan Y, Wang L, Qiu Z, Liu M, Chen Y, Wu L, Li Y, Ma X, Liu M, Li D (2014) CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos. Nat Protoc 9:2493–2512CrossRefPubMedGoogle Scholar
  23. 23.
    Chapman KM, Medrano GA, Jaichander P, Chaudhary J, Waits AE, Nobrega MA, Hotaling JM, Ober C, Hamra FK (2015) Targeted germline modifications in rats using CRISPR/Cas9 and spermatogonial stem cells. Cell Rep 10:1828–1835CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Miao J, Guo D, Zhang J, Huang Q, Qin G, Zhang X, Wan J, Gu H, Qu LJ (2013) Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res 23:1233–1236CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233–239CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Yosef I, Manor M, Kiro R, Qimron U (2015) Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria. Proc Natl Acad Sci USA 112:7267–7272CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Dickinson DJ, Ward JD, Reiner DJ, Goldstein B (2013) Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat Methods 10:1028–1034CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Friedland AE, Tzur YB, Esvelt KM, Colaiacovo MP, Church GM, Calarco JA (2013) Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods 10:741–743CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227–229CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227–229CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Jao LE, Wente SR, Chen W (2013) Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci USA 110:13904–13909CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    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 24:372–375CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Zhou X, Xin J, Fan N, Zou Q, Huang J, Ouyang Z, Zhao Y, Zhao B, Liu Z, Lai S et al (2015) Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cell Mol Life Sci 72:1175–1184CrossRefPubMedGoogle Scholar
  34. 34.
    Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W et al (2014) Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156:836–843CrossRefPubMedGoogle Scholar
  35. 35.
    Wakayama S, Kohda T, Obokata H, Tokoro M, Li C, Terashita Y, Mizutani E, Nguyen VT, Kishigami S, Ishino F, Wakayama T (2013) Successful serial recloning in the mouse over multiple generations. Cell Stem Cell 12:293–297CrossRefPubMedGoogle Scholar
  36. 36.
    Merkle FT, Neuhausser WM, Santos D, Valen E, Gagnon JA, Maas K, Sandoe J, Schier AF, Eggan K (2015) Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus. Cell Rep 11:875–883CrossRefPubMedGoogle Scholar
  37. 37.
    Kim Y, Kweon J, Kim A, Chon JK, Yoo JY, Kim HJ, Kim S, Lee C, Jeong E, Chung E et al (2013) A library of TAL effector nucleases spanning the human genome. Nat Biotechnol 31:251–258CrossRefPubMedGoogle Scholar
  38. 38.
    Christian ML, Demorest ZL, Starker CG, Osborn MJ, Nyquist MD, Zhang Y, Carlson DF, Bradley P, Bogdanove AJ, Voytas DF (2012) Targeting G with TAL effectors: a comparison of activities of TALENs constructed with NN and NK repeat variable di-residues. PLoS One 7:e45383CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu JL, Gao C (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31:686–688CrossRefPubMedGoogle Scholar
  40. 40.
    Cho SW, Kim S, Kim JM, Kim JS (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31:230–232CrossRefPubMedGoogle Scholar
  41. 41.
    Duda K, Lonowski LA, Kofoed-Nielsen M, Ibarra A, Delay CM, Kang Q, Yang Z, Pruett-Miller SM, Bennett EP, Wandall HH et al (2014) High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs. Nucleic Acids Res 42:e84CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Feng Y, Zhang S, Huang X (2014) A robust TALENs system for highly efficient mammalian genome editing. Sci Rep 4:3632PubMedPubMedCentralGoogle Scholar
  43. 43.
    Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, Wang N, Lee G, Bartsevich VV, Lee YL et al (2008) Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 26:808–816CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Ren C, Xu K, Liu Z, Shen J, Han F, Chen Z, Zhang Z (2015) Dual-reporter surrogate systems for efficient enrichment of genetically modified cells. Cell Mol Life Sci 72:2763–2772CrossRefPubMedGoogle Scholar
  45. 45.
    Ramakrishna S, Cho SW, Kim S, Song M, Gopalappa R, Kim JS, Kim H (2014) Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations. Nat Commun 5:3378CrossRefPubMedGoogle Scholar
  46. 46.
    Lim S, Wang Y, Yu X, Huang Y, Featherstone MS, Sampath K (2013) A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases. Genome Biol 14:R69CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Lieber MR (2010) The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 79:181–211CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Li X, Heyer WD (2008) Homologous recombination in DNA repair and DNA damage tolerance. Cell Res 18:99–113CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Kim H, Um E, Cho SR, Jung C, Kim H, Kim JS (2011) Surrogate reporters for enrichment of cells with nuclease-induced mutations. Nat Methods 8:941–943CrossRefPubMedGoogle Scholar
  50. 50.
    Liu Y, Lv X, Tan R, Liu T, Chen T, Li M, Liu Y, Nie F, Wang X, Zhou P et al (2014) A modified TALEN-based strategy for rapidly and efficiently generating knockout mice for kidney development studies. PLoS One 9:e84893CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, Schmidt C, Baller JA, Somia NV, Bogdanove AJ, Voytas DF (2011) Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39:e82CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Luo Y, Lin L, Bolund L, Sorensen CB (2014) Efficient construction of rAAV-based gene targeting vectors by Golden Gate cloning. Biotechniques 56:263–268PubMedGoogle Scholar
  53. 53.
    Szczepek M, Brondani V, Buchel J, Serrano L, Segal DJ, Cathomen T (2007) Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol 25:786–793CrossRefPubMedGoogle Scholar
  54. 54.
    Ochiai H, Fujita K, Suzuki K, Nishikawa M, Shibata T, Sakamoto N, Yamamoto T (2010) Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases. Genes Cells 15:875–885PubMedGoogle Scholar
  55. 55.
    Kim HK, Kaang BK (1998) Truncated green fluorescent protein mutants and their expression in Aplysia neurons. Brain Res Bull 47:35–41CrossRefPubMedGoogle Scholar
  56. 56.
    Luo Y, Lin L, Bolund L, Jensen TG, Sorensen CB (2012) Genetically modified pigs for biomedical research. J Inherit Metab Dis 35:695–713CrossRefPubMedGoogle Scholar
  57. 57.
    Holm IE, Alstrup AK, Luo Y (2015) Genetically modified pig models for neurodegenerative disorders. J Pathol. doi: 10.1002/path.4654 PubMedGoogle Scholar
  58. 58.
    Zhang X, Cheng B, Gong H, Li C, Chen H, Zheng L, Huang K (2011) Porcine islet amyloid polypeptide fragments are refractory to amyloid formation. FEBS Lett 585:71–77CrossRefPubMedGoogle Scholar
  59. 59.
    Sung YH, Baek IJ, Kim DH, Jeon J, Lee J, Lee K, Jeong D, Kim JS, Lee HW (2013) Knockout mice created by TALEN-mediated gene targeting. Nat Biotechnol 31:23–24CrossRefPubMedGoogle Scholar
  60. 60.
    Xin J, Yang H, Fan N, Zhao B, Ouyang Z, Liu Z, Zhao Y, Li X, Song J, Yang Y et al (2013) Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs. PLoS One 8:e84250CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Blechingberg J, Luo Y, Bolund L, Damgaard CK, Nielsen AL (2012) Gene expression responses to FUS, EWS, and TAF15 reduction and stress granule sequestration analyses identifies FET-protein non-redundant functions. PLoS One 7:e46251CrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    Sigal A, Danon T, Cohen A, Milo R, Geva-Zatorsky N, Lustig G, Liron Y, Alon U, Perzov N (2007) Generation of a fluorescently labeled endogenous protein library in living human cells. Nat Protoc 2:1515–1527CrossRefPubMedGoogle Scholar
  63. 63.
    Zhou D, Ren JX, Ryan TM, Higgins NP, Townes TM (2004) Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts. Nucleic Acids Res 32:e128CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL (2015) Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol 33:538–542CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
    Gratz SJ, Ukken FP, Rubinstein CD, Thiede G, Donohue LK, Cummings AM, O’Connor-Giles KM (2014) Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics 196:961–971CrossRefPubMedPubMedCentralGoogle Scholar
  66. 66.
    Auer TO, Duroure K, De Cian A, Concordet JP, Del Bene F (2014) Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res 24:142–153CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    Port F, Chen HM, Lee T, Bullock SL (2014) Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci USA 111:E2967–E2976CrossRefPubMedPubMedCentralGoogle Scholar
  68. 68.
    Mignone F, Gissi C, Liuni S, Pesole G (2002) Untranslated regions of mRNAs. Genome Biol 3(REVIEWS000):4Google Scholar
  69. 69.
    Zou L, Luo Y, Chen M, Wang G, Ding M, Petersen CC, Kang R, Dagnaes-Hansen F, Zeng Y, Lv N et al (2013) A simple method for deriving functional MSCs and applied for osteogenesis in 3D scaffolds. Sci Rep 3:2243PubMedPubMedCentralGoogle Scholar
  70. 70.
    Siddle K (2011) Signalling by insulin and IGF receptors: supporting acts and new players. J Mol Endocrinol 47:R1–R10CrossRefPubMedGoogle Scholar
  71. 71.
    Brandl C, Ortiz O, Rottig B, Wefers B, Wurst W, Kuhn R (2015) Creation of targeted genomic deletions using TALEN or CRISPR/Cas nuclease pairs in one-cell mouse embryos. FEBS Open Bio 5:26–35CrossRefPubMedGoogle Scholar
  72. 72.
    Levenson AS, Jordan VC (1997) MCF-7: the first hormone-responsive breast cancer cell line. Cancer Res 57:3071–3078PubMedGoogle Scholar
  73. 73.
    Norwood LE, Grade SK, Cryderman DE, Hines KA, Furiasse N, Toro R, Li Y, Dhasarathy A, Kladde MP, Hendrix MJ et al (2004) Conserved properties of HP1(Hsalpha). Gene 336:37–46CrossRefPubMedGoogle Scholar
  74. 74.
    Kirschmann DA, Lininger RA, Gardner LM, Seftor EA, Odero VA, Ainsztein AM, Earnshaw WC, Wallrath LL, Hendrix MJ (2000) Down-regulation of HP1Hsalpha expression is associated with the metastatic phenotype in breast cancer. Cancer Res 60:3359–3363PubMedGoogle Scholar
  75. 75.
    Ceccaldi R, Rondinelli B, D’Andrea AD (2015) Repair pathway choices and consequences at the double-strand break. Trends Cell Biol S0962–8924(15):00142–00147Google Scholar
  76. 76.
    Bae S, Park J, Kim JS (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30:1473–1475CrossRefPubMedPubMedCentralGoogle Scholar
  77. 77.
    Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E (2014) CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res 42:W401–W407CrossRefPubMedPubMedCentralGoogle Scholar
  78. 78.
    Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32:279–284CrossRefPubMedPubMedCentralGoogle Scholar
  79. 79.
    Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31:839–843CrossRefPubMedPubMedCentralGoogle Scholar
  80. 80.
    Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, Kim JS (2014) Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 24:132–141CrossRefPubMedPubMedCentralGoogle Scholar
  81. 81.
    Kim E, Kim S, Kim DH, Choi BS, Choi IY, Kim JS (2012) Precision genome engineering with programmable DNA-nicking enzymes. Genome Res 22:1327–1333CrossRefPubMedPubMedCentralGoogle Scholar
  82. 82.
    Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31:833–838CrossRefPubMedPubMedCentralGoogle Scholar
  83. 83.
    Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31:822–826CrossRefPubMedPubMedCentralGoogle Scholar
  84. 84.
    Li Y, Park AI, Mou H, Colpan C, Bizhanova A, Akama-Garren E, Joshi N, Hendrickson EA, Feldser D, Yin H et al (2015) A versatile reporter system for CRISPR-mediated chromosomal rearrangements. Genome Biol 16:111CrossRefPubMedPubMedCentralGoogle Scholar
  85. 85.
    Shrivastav M, De Haro LP, Nickoloff JA (2008) Regulation of DNA double-strand break repair pathway choice. Cell Res 18:134–147CrossRefPubMedGoogle Scholar
  86. 86.
    Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X, Skarnes WC (2014) Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods 11:399–402CrossRefPubMedGoogle Scholar
  87. 87.
    Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol 32:569–576CrossRefPubMedPubMedCentralGoogle Scholar
  88. 88.
    Sung P, Klein H (2006) Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol 7:739–750CrossRefPubMedGoogle Scholar
  89. 89.
    Voit RA, Hendel A, Pruett-Miller SM, Porteus MH (2014) Nuclease-mediated gene editing by homologous recombination of the human globin locus. Nucleic Acids Res 42:1365–1378CrossRefPubMedGoogle Scholar
  90. 90.
    Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872CrossRefPubMedGoogle Scholar
  91. 91.
    Lorson MA, Spate LD, Samuel MS, Murphy CN, Lorson CL, Prather RS, Wells KD (2011) Disruption of the survival motor neuron (SMN) gene in pigs using ssDNA. Transgenic Res 20:1293–1304CrossRefPubMedPubMedCentralGoogle Scholar
  92. 92.
    Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN et al (2002) Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295:1089–1092CrossRefPubMedGoogle Scholar
  93. 93.
    Williams SH, Sahota V, Palmai-Pallag T, Tebbutt SJ, Walker J, Harris A (2003) Evaluation of gene targeting by homologous recombination in ovine somatic cells. Mol Reprod Dev 66:115–125CrossRefPubMedGoogle Scholar
  94. 94.
    Du Y, Kragh PM, Zhang Y, Li J, Schmidt M, Bogh IB, Zhang X, Purup S, Jorgensen AL, Pedersen AM et al (2007) Piglets born from handmade cloning, an innovative cloning method without micromanipulation. Theriogenology 68:1104–1110CrossRefPubMedGoogle Scholar
  95. 95.
    Luo Y, Lin L, Golas M, Sørensen CB, Bolund L (2015) Targeted porcine genome engineering with TALENs. In: Li X-Q, Jensen TG (eds) Somatic genome manipulation: advances, methods and applications, 1st edn. Springer, BerlinGoogle Scholar
  96. 96.
    Luo Y, Bolund L, Sorensen CB (2012) Pig gene knockout by rAAV-mediated homologous recombination: comparison of BRCA1 gene knockout efficiency in Yucatan and Gottingen fibroblasts with slightly different target sequences. Transgenic Res 21:671–676CrossRefPubMedGoogle Scholar
  97. 97.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing 2016

Authors and Affiliations

  • Yan Zhou
    • 1
  • Yong Liu
    • 1
  • Dianna Hussmann
    • 1
  • Peter Brøgger
    • 1
  • Rasha Abdelkadhem Al-Saaidi
    • 2
  • Shuang Tan
    • 1
    • 3
  • Lin Lin
    • 1
  • Trine Skov Petersen
    • 1
  • Guang Qian Zhou
    • 3
  • Peter Bross
    • 2
  • Lars Aagaard
    • 1
  • Tino Klein
    • 5
  • Sif Groth Rønn
    • 6
  • Henrik Duelund Pedersen
    • 6
  • Lars Bolund
    • 1
    • 4
    • 7
  • Anders Lade Nielsen
    • 1
  • Charlotte Brandt Sørensen
    • 2
  • Yonglun Luo
    • 1
    • 6
    • 7
  1. 1.Department of BiomedicineAarhus UniversityAarhus CDenmark
  2. 2.Research Unit for Molecular Medicine, Department of Clinical MedicineAarhus University and University HospitalAarhus NDenmark
  3. 3.Shenzhen Key Laboratory for Anti-aging and Regenerative Medicine, Health Science CenterShenzhen UniversityShenzhenChina
  4. 4.BGI-ShenzhenShenzhenChina
  5. 5.Department of HistologyGubra A/SHørsholmDenmark
  6. 6.Department of Incretin and Obesity ResearchNovo Nordisk A/SMåløvDenmark
  7. 7.The Danish Regenerative Engineering Alliance for Medicine (DREAM)Aarhus UniversityAarhusDenmark

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