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Beyond editing to writing large genomes

Abstract

Recent exponential advances in genome sequencing and engineering technologies have enabled an unprecedented level of interrogation into the impact of DNA variation (genotype) on cellular function (phenotype). Furthermore, these advances have also prompted realistic discussion of writing and radically re-writing complex genomes. In this Perspective, we detail the motivation for large-scale engineering, discuss the progress made from such projects in bacteria and yeast and describe how various genome-engineering technologies will contribute to this effort. Finally, we describe the features of an ideal platform and provide a roadmap to facilitate the efficient writing of large genomes.

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Figure 1: Timeline of gene targeting.
Figure 2: Potential applications of large-scale genome engineering of different organisms.
Figure 3: DNA-editing nanomachines.
Figure 4: Two main approaches to large-scale genome engineering in bacteria and yeast.
Figure 5: A roadmap to building large genomes using a combination of de novo synthesis and genome editing.

References

  1. Kosuri, S. & Church, G. M. Large-scale de novo DNA synthesis: technologies and applications. Nat. Methods 11, 499–507 (2014).

    CAS  PubMed  Google Scholar 

  2. Reuter, J. A., Spacek, D. V. & Snyder, M. P. High-throughput sequencing technologies. Mol. Cell 58, 586–597 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Goodwin, S., McPherson, J. D. & McCombie, W. R. Coming of age: ten years of next-generation sequencing technologies. Nat. Rev. Genet. 17, 333–351 (2016).

    CAS  PubMed  Google Scholar 

  4. Smithies, O., Gregg, R. G., Boggs, S. S., Koralewski, M. A. & Kucherlapati, R. S. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature 317, 230–234 (1985).

    CAS  PubMed  Google Scholar 

  5. DeWitt, M. A. et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci. Transl Med. 8, 360ra134 (2016).

    PubMed  PubMed Central  Google Scholar 

  6. Byrne, S. M., Ortiz, L., Mali, P., Aach, J. & Church, G. M. Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells. Nucleic Acids Res. 43, e21 (2015).

    PubMed  Google Scholar 

  7. Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Wang, H. H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Boeke, J. D. et al. The Genome Project-Write. Science 353, 126–127 (2016).

    CAS  PubMed  Google Scholar 

  10. Robertson, D. E. et al. A new dawn for industrial photosynthesis. Photosynth. Res. 107, 269–277 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Salhi, A. et al. DESM: portal for microbial knowledge exploration systems. Nucleic Acids Res. 44, D624–D633 (2016).

    CAS  PubMed  Google Scholar 

  12. Fischer, C. R., Klein-Marcuschamer, D. & Stephanopoulos, G. Selection and optimization of microbial hosts for biofuels production. Metab. Eng. 10, 295–304 (2008).

    CAS  PubMed  Google Scholar 

  13. Isaacs, F. J. et al. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333, 348–353 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Lajoie, M. J. et al. Genomically recoded organisms expand biological functions. Science 342, 357–360 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Welter, D. et al. The NHGRI GWAS Catalog, a curated resource of SNP-trait associations. Nucleic Acids Res. 42, D1001–D1006 (2014).

    CAS  PubMed  Google Scholar 

  16. Wang, G. et al. Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nat. Med. 20, 616–623 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Freedman, B. S. et al. Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids. Nat. Commun. 6, 8715 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Drost, J. et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521, 43–47 (2015).

    CAS  PubMed  Google Scholar 

  19. Matano, M. et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat. Med. 21, 256–262 (2015).

    CAS  PubMed  Google Scholar 

  20. Schwank, G. et al. Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell 13, 653–658 (2013).

    CAS  PubMed  Google Scholar 

  21. Liu, J. et al. CRISPR/Cas9 facilitates investigation of neural circuit disease using human iPSCs: mechanism of epilepsy caused by an SCN1A loss-of-function mutation. Transl Psychiatry 6, e703 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Soldner, F. et al. Parkinson-associated risk variant in distal enhancer of α-synuclein modulates target gene expression. Nature 533, 95–99 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Chan, L. Y., Kosuri, S. & Endy, D. Refactoring bacteriophage T7. Mol. Syst. Biol. 1, 2005.0018 (2005).

    PubMed  PubMed Central  Google Scholar 

  24. Mandell, D. J. et al. Biocontainment of genetically modified organisms by synthetic protein design. Nature 518, 55–60 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Rovner, A. J. et al. Recoded organisms engineered to depend on synthetic amino acids. Nature 518, 89–93 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).

    PubMed  PubMed Central  Google Scholar 

  27. Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Zhang, Y., Buchholz, F., Muyrers, J. P. & Stewart, A. F. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20, 123–128 (1998).

    CAS  PubMed  Google Scholar 

  30. Yu, D. et al. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl Acad. Sci. USA 97, 5978–5983 (2000).

    CAS  PubMed  Google Scholar 

  31. Lacroix, B. & Citovsky, V. Transfer of DNA from bacteria to eukaryotes. mBio 7, e00863–16 (2016).

    PubMed  PubMed Central  Google Scholar 

  32. Xu, K., Stewart, A. F. & Porter, A. C. G. Stimulation of oligonucleotide-directed gene correction by Redβ expression and MSH2 depletion in human HT1080 cells. Mol. Cells 38, 33–39 (2015).

    PubMed  Google Scholar 

  33. Rios, X. et al. Stable gene targeting in human cells using single-strand oligonucleotides with modified bases. PLoS ONE 7, e36697 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Igoucheva, O., Alexeev, V. & Yoon, K. Targeted gene correction by small single-stranded oligonucleotides in mammalian cells. Gene Ther. 8, 391–399 (2001).

    CAS  PubMed  Google Scholar 

  35. Aarts, M. & te Riele, H. Subtle gene modification in mouse ES cells: evidence for incorporation of unmodified oligonucleotides without induction of DNA damage. Nucleic Acids Res. 38, 6956–6967 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Quadros, R. M. et al. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol. 18, 92 (2017).

    PubMed  PubMed Central  Google Scholar 

  37. Swarts, D. C. et al. DNA-guided DNA interference by a prokaryotic Argonaute. Nature 507, 258–261 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Gao, F., Shen, X. Z., Jiang, F., Wu, Y. & Han, C. DNA-guided genome editing using the Natronobacterium gregoryi Argonaute. Nat. Biotechnol. 34, 768–773 (2016).

    CAS  PubMed  Google Scholar 

  39. Lee, S. H. et al. Failure to detect DNA-guided genome editing using Natronobacterium gregoryi Argonaute. Nat. Biotechnol. 35, 17–18 (2016).

    PubMed  PubMed Central  Google Scholar 

  40. Burgess, S. et al. Questions about NgAgo. Protein Cell 7, 913–915 (2016).

    PubMed  PubMed Central  Google Scholar 

  41. Javidi-Parsijani, P. et al. No evidence of genome editing activity from Natronobacterium gregoryi Argonaute (NgAgo) in human cells. PLoS ONE 12, e0177444 (2017).

    PubMed  PubMed Central  Google Scholar 

  42. Kim, H. & Kim, J.-S. A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15, 321–334 (2014).

    CAS  PubMed  Google Scholar 

  43. Chandrasegaran, S. & Carroll, D. Origins of programmable nucleases for genome engineering. J. Mol. Biol. 428, 963–989 (2016).

    CAS  PubMed  Google Scholar 

  44. Chevalier, B. S. & Stoddard, B. L. Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility. Nucleic Acids Res. 29, 3757–3774 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Smith, J. et al. A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. Nucleic Acids Res. 34, e149 (2006).

    PubMed  PubMed Central  Google Scholar 

  46. Chevalier, B. S. et al. Design, activity, and structure of a highly specific artificial endonuclease. Mol. Cell 10, 895–905 (2002).

    CAS  PubMed  Google Scholar 

  47. Boissel, S. et al. megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering. Nucleic Acids Res. 42, 2591–2601 (2014).

    CAS  PubMed  Google Scholar 

  48. Wolfs, J. M. et al. MegaTevs: single-chain dual nucleases for efficient gene disruption. Nucleic Acids Res. 42, 8816–8829 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Birling, M.-C., Gofflot, F. & Warot, X. Site-specific recombinases for manipulation of the mouse genome. Methods Mol. Biol. 561, 245–263 (2009).

    CAS  PubMed  Google Scholar 

  50. Sauer, B. & Henderson, N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc. Natl Acad. Sci. USA 85, 5166–5170 (1988).

    CAS  PubMed  Google Scholar 

  51. Turan, S. & Bode, J. Site-specific recombinases: from tag-and-target-to tag-and-exchange-based genomic modifications. FASEB J. 25, 4088–4107 (2011).

    CAS  PubMed  Google Scholar 

  52. Buchholz, F. & Stewart, A. F. Alteration of Cre recombinase site specificity by substrate-linked protein evolution. Nat. Biotechnol. 19, 1047–1052 (2001).

    CAS  PubMed  Google Scholar 

  53. Eroshenko, N. & Church, G. M. Mutants of Cre recombinase with improved accuracy. Nat. Commun. 4, 2509 (2013).

    PubMed  PubMed Central  Google Scholar 

  54. Santoro, S. W. & Schultz, P. G. Directed evolution of the site specificity of Cre recombinase. Proc. Natl Acad. Sci. USA 99, 4185–4190 (2002).

    CAS  PubMed  Google Scholar 

  55. Saraf-Levy, T. et al. Site-specific recombination of asymmetric lox sites mediated by a heterotetrameric Cre recombinase complex. Bioorg. Med. Chem. 14, 3081–3089 (2006).

    CAS  PubMed  Google Scholar 

  56. Gaj, T. et al. Enhancing the specificity of recombinase-mediated genome engineering through dimer interface redesign. J. Am. Chem. Soc. 136, 5047–5056 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Wallen, M. C., Gaj, T. & Barbas, C. F. Redesigning recombinase specificity for safe harbor sites in the human genome. PLoS ONE 10, e0139123 (2015).

    PubMed  PubMed Central  Google Scholar 

  58. Mastroianni, M. et al. Group II intron-based gene targeting reactions in eukaryotes. PLoS ONE 3, e3121 (2008).

    PubMed  PubMed Central  Google Scholar 

  59. Guo, H. et al. Group II introns designed to insert into therapeutically relevant DNA target sites in human cells. Science 289, 452–457 (2000).

    CAS  PubMed  Google Scholar 

  60. Shalem, O., Sanjana, N. E. & Zhang, F. High-throughput functional genomics using CRISPR-Cas9. Nat. Rev. Genet. 16, 299–311 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Tsai, S. Q. & Joung, J. K. Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat. Rev. Genet. 17, 300–312 (2016).

    CAS  PubMed  Google Scholar 

  62. Komor, A. C., Badran, A. H. & Liu, D. R. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell 168, 20–36 (2017).

    CAS  PubMed  Google Scholar 

  63. Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).

    PubMed  Google Scholar 

  67. Yang, L. et al. Engineering and optimising deaminase fusions for genome editing. Nat. Commun. 7, 13330 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Fulco, C. P. et al. Systematic mapping of functional enhancer-promoter connections with CRISPR interference. Science 354, 769–773 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Canver, M. C. et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527, 192–197 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. Yang, L. et al. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science 350, 1101–1104 (2015).

    CAS  PubMed  Google Scholar 

  71. Haimovich, A. D., Muir, P. & Isaacs, F. J. Genomes by design. Nat. Rev. Genet. 16, 501–516 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Smith, H. O., Hutchison, C. A., Pfannkoch, C. & Venter, J. C. Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides. Proc. Natl Acad. Sci. USA 100, 15440–15445 (2003).

    CAS  PubMed  Google Scholar 

  73. Gibson, D. G. et al. Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215–1220 (2008).

    CAS  PubMed  Google Scholar 

  74. Gibson, D. G. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52–56 (2010).

    CAS  PubMed  Google Scholar 

  75. Hutchison, C. A. et al. Design and synthesis of a minimal bacterial genome. Science 351, aad6253 (2016).

    PubMed  Google Scholar 

  76. Lartigue, C. et al. Creating bacterial strains from genomes that have been cloned and engineered in yeast. Science 325, 1693–1696 (2009).

    CAS  PubMed  Google Scholar 

  77. Ostrov, N. et al. Design, synthesis, and testing toward a 57-codon genome. Science 353, 819–822 (2016).

    CAS  PubMed  Google Scholar 

  78. Dymond, J. S. et al. Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature 477, 471–476 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Annaluru, N. et al. Total synthesis of a functional designer eukaryotic chromosome. Science 344, 55–58 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Richardson, S. M. et al. Design of a synthetic yeast genome. Science 355, 1040–1044 (2017).

    CAS  PubMed  Google Scholar 

  81. Shen, Y. et al. Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome. Science 355, eaaf4791 (2017).

    PubMed  PubMed Central  Google Scholar 

  82. Xie, Z.-X. et al. 'Perfect' designer chromosome V and behavior of a ring derivative. Science 355, eaaf4704 (2017).

    PubMed  Google Scholar 

  83. Zhang, W. et al. Engineering the ribosomal DNA in a megabase synthetic chromosome. Science 355, eaaf3981 (2017).

    PubMed  Google Scholar 

  84. Mitchell, L. A. et al. Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond. Science 355, eaaf4831 (2017).

    PubMed  Google Scholar 

  85. Shendure, J. et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728–1732 (2005).

    CAS  PubMed  Google Scholar 

  86. Tian, J. et al. Accurate multiplex gene synthesis from programmable DNA microchips. Nature 432, 1050–1054 (2004).

    CAS  PubMed  Google Scholar 

  87. Gordon, D. et al. Long-read sequence assembly of the gorilla genome. Science 352, aae0344 (2016).

    PubMed  PubMed Central  Google Scholar 

  88. Loose, M., Malla, S. & Stout, M. Real-time selective sequencing using nanopore technology. Nat. Methods 13, 751–754 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. Szalay, T. & Golovchenko, J. A. De novo sequencing and variant calling with nanopores using PoreSeq. Nat. Biotechnol. 33, 1087–1091 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. Goodwin, S. et al. Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome. Genome Res. 25, 1750–1756 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. Fuller, C. W. et al. Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array. Proc. Natl Acad. Sci. USA 113, 5233–5238 (2016).

    CAS  PubMed  Google Scholar 

  92. Beliveau, B. J. et al. Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes. Nat. Commun. 6, 7147 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

    CAS  PubMed  Google Scholar 

  95. Frock, R. L. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. 33, 179–186 (2015).

    CAS  PubMed  Google Scholar 

  96. Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015).

    CAS  PubMed  Google Scholar 

  97. Lensing, S. V. et al. DSBCapture: in situ capture and sequencing of DNA breaks. Nat. Methods 13, 855–857 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  98. Osoegawa, K. et al. A bacterial artificial chromosome library for sequencing the complete human genome. Genome Res. 11, 483–496 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  99. Straub, C., Granger, A. J., Saulnier, J. L. & Sabatini, B. L. CRISPR/Cas9-mediated gene knock-down in post-mitotic neurons. PLoS ONE 9, e105584 (2014).

    PubMed  PubMed Central  Google Scholar 

  100. Agah, R. et al. Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo. J. Clin. Invest. 100, 169–179 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  101. Suzuki, K. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144–149 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. Pinder, J., Salsman, J. & Dellaire, G. Nuclear domain 'knock-in' screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing. Nucleic Acids Res. 43, 9379–9392 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  103. Chu, V. T. et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548 (2015).

    CAS  PubMed  Google Scholar 

  104. Maruyama, T. et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 33, 538–542 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  105. Adamson, B., Smogorzewska, A., Sigoillot, F. D., King, R. W. & Elledge, S. J. A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response. Nat. Cell Biol. 14, 318–328 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. Agudelo, D. et al. Marker-free coselection for CRISPR-driven genome editing in human cells. Nat. Methods 14, 615–620 (2017).

    CAS  PubMed  Google Scholar 

  107. Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. eLife 3, e04766 (2014).

    PubMed  PubMed Central  Google Scholar 

  108. He, X. et al. Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair. Nucleic Acids Res. 44, e85 (2016).

    PubMed  PubMed Central  Google Scholar 

  109. Maresca, M., Lin, V. G., Guo, N. & Yang, Y. Obligate ligation-gated recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining. Genome Res. 23, 539–546 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  110. Yang, Y. & Seed, B. Site-specific gene targeting in mouse embryonic stem cells with intact bacterial artificial chromosomes. Nat. Biotechnol. 21, 447–451 (2003).

    CAS  PubMed  Google Scholar 

  111. Bradley, A. et al. The mammalian gene function resource: the International Knockout Mouse Consortium. Mamm. Genome 23, 580–586 (2012).

    PubMed  PubMed Central  Google Scholar 

  112. Ordovás, L. et al. Efficient recombinase-mediated cassette exchange in hPSCs to study the hepatocyte lineage reveals AAVS1 locus-mediated transgene inhibition. Stem Cell Rep. 5, 918–931 (2015).

    Google Scholar 

  113. Voziyanova, E. et al. Efficient Flp-Int HK022 dual RMCE in mammalian cells. Nucleic Acids Res. 41, e125 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  114. Osterwalder, M. et al. Dual RMCE for efficient re-engineering of mouse mutant alleles. Nat. Methods 7, 893–895 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  115. Zhang, X., Koolhaas, W. H. & Schnorrer, F. A versatile two-step CRISPR- and RMCE-based strategy for efficient genome engineering in Drosophila. G3 (Bethesda) 4, 2409–2418 (2014).

    Google Scholar 

  116. Bosley, K. S. et al. CRISPR germline engineering — the community speaks. Nat. Biotechnol. 33, 478–486 (2015).

    CAS  PubMed  Google Scholar 

  117. Baltimore, D. et al. Biotechnology. A prudent path forward for genomic engineering and germline gene modification. Science 348, 36–38 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  118. Church, G. Perspective: encourage the innovators. Nature 528, S7 (2015).

    CAS  PubMed  Google Scholar 

  119. Doudna, J. Perspective: embryo editing needs scrutiny. Nature 528, S6 (2015).

    CAS  PubMed  Google Scholar 

  120. Schandera, J. & Mackey, T. K. Mitochondrial replacement techniques: divergence in global policy. Trends Genet. 32, 385–390 (2016).

    CAS  PubMed  Google Scholar 

  121. Li, L. & Blankenstein, T. Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast-embryonic stem cell fusion. Nat. Protoc. 8, 1567–1582 (2013).

    PubMed  Google Scholar 

  122. Tomizuka, K. et al. Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies. Proc. Natl Acad. Sci. USA 97, 722–727 (2000).

    CAS  PubMed  Google Scholar 

  123. DiCarlo, J. E. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 41, 4336–4343 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. EauClaire, S. F., Zhang, J., Rivera, C. G. & Huang, L. L. Combinatorial metabolic pathway assembly in the yeast genome with RNA-guided Cas9. J. Ind. Microbiol. Biotechnol. 43, 1001–1015 (2016).

    CAS  PubMed  Google Scholar 

  125. Wang, S.-Z., Liu, B.-H., Tao, H. W., Xia, K. & Zhang, L. I. A genetic strategy for stochastic gene activation with regulated sparseness (STARS). PLoS ONE 4, e4200 (2009).

    PubMed  PubMed Central  Google Scholar 

  126. Gouble, A. et al. Efficient in toto targeted recombination in mouse liver by meganuclease-induced double-strand break. J. Gene Med. 8, 616–622 (2006).

    CAS  PubMed  Google Scholar 

  127. Gellhaus, K., Cornu, T. I., Heilbronn, R. & Cathomen, T. Fate of recombinant adeno-associated viral vector genomes during DNA double-strand break-induced gene targeting in human cells. Hum. Gene Ther. 21, 543–553 (2010).

    CAS  PubMed  Google Scholar 

  128. Robert, M.-A. et al. Efficacy and site-specificity of adenoviral vector integration mediated by the phage φC31 integrase. Hum. Gene Ther. Methods 23, 393–407 (2012).

    CAS  PubMed  Google Scholar 

  129. Ou, H. et al. A highly efficient site-specific integration strategy using combination of homologous recombination and the ΦC31 integrase. J. Biotechnol. 167, 427–432 (2013).

    CAS  PubMed  Google Scholar 

  130. Coluccio, A. et al. Targeted gene addition in human epithelial stem cells by zinc-finger nuclease-mediated homologous recombination. Mol. Ther. 21, 1695–1704 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  131. Beaton, B. P. et al. Inclusion of homologous DNA in nuclease-mediated gene targeting facilitates a higher incidence of bi-allelically modified cells. Xenotransplantation 22, 379–390 (2015).

    PubMed  PubMed Central  Google Scholar 

  132. Nakade, S. et al. Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat. Commun. 5, 5560 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  133. Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L. & Corn, J. E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat. Biotechnol. 34, 339–344 (2016).

    CAS  PubMed  Google Scholar 

  134. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  135. Puchta, H., Dujon, B. & Hohn, B. Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease. Nucleic Acids Res. 21, 5034–5040 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  136. Groth, A. C., Olivares, E. C., Thyagarajan, B. & Calos, M. P. A phage integrase directs efficient site-specific integration in human cells. Proc. Natl Acad. Sci. USA 97, 5995–6000 (2000).

    CAS  PubMed  Google Scholar 

  137. Bibikova, M. et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol. Cell. Biol. 21, 289–297 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  138. Rouet, P., Smih, F. & Jasin, M. Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc. Natl Acad. Sci. USA 91, 6064–6068 (1994).

    CAS  PubMed  Google Scholar 

  139. Liu, P., Jenkins, N. A. & Copeland, N. G. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res. 13, 476–484 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  140. Testa, G. et al. Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nat. Biotechnol. 21, 443–447 (2003).

    CAS  PubMed  Google Scholar 

  141. Epinat, J.-C. et al. A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells. Nucleic Acids Res. 31, 2952–2962 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  142. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  143. Mercer, A. C., Gaj, T., Fuller, R. P. & Barbas, C. F. Chimeric TALE recombinases with programmable DNA sequence specificity. Nucleic Acids Res. 40, 11163–11172 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  144. Gaj, T., Mercer, A. C., Sirk, S. J., Smith, H. L. & Barbas, C. F. A comprehensive approach to zinc-finger recombinase customization enables genomic targeting in human cells. Nucleic Acids Res. 41, 3937–3946 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  145. Owens, J. B. et al. Transcription activator like effector (TALE)-directed piggyBac transposition in human cells. Nucleic Acids Res. 41, 9197–9207 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  146. Tsai, S. Q. et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32, 569–576 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  147. Guilinger, J. P., Thompson, D. B. & Liu, D. R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32, 577–582 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  148. Pettersen, E. F. et al. UCSF Chimera — a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  PubMed  Google Scholar 

  149. Zhang, J., McCabe, K. A. & Bell, C. E. Crystal structures of λ exonuclease in complex with DNA suggest an electrostatic ratchet mechanism for processivity. Proc. Natl Acad. Sci. USA. 108, 11872–11877 (2011).

    CAS  PubMed  Google Scholar 

  150. Chen, Z., Yang, H. & Pavletich, N. P. Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures. Nature 453, 489–484 (2008).

    CAS  PubMed  Google Scholar 

  151. Sheng, G. et al. Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage. Proc. Natl Acad. Sci. USA 111, 652–657 (2014).

    CAS  PubMed  Google Scholar 

  152. Muñoz, I. G. et al. Molecular basis of engineered meganuclease targeting of the endogenous human RAG1 locus. Nucleic Acids Res. 39, 729–743 (2011).

    PubMed  Google Scholar 

  153. Gopaul, D. N., Guo, F. & Van Duyne, G. D. Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination. EMBO J. 17, 4175–4187 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  154. Peisach, E. & Pabo, C. O. Constraints for zinc finger linker design as inferred from X-ray crystal structure of tandem Zif268-DNA complexes. J. Mol. Biol. 330, 1–7 (2003).

    CAS  PubMed  Google Scholar 

  155. Mak, A. N.-S., Bradley, P., Cernadas, R. A., Bogdanove, A. J. & Stoddard, B. L. The crystal structure of TAL effector PthXo1 bound to its DNA target. Science 335, 716–719 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  156. Toor, N., Keating, K. S., Taylor, S. D. & Pyle, A. M. Crystal structure of a self-spliced group II intron. Science 320, 77–82 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  157. Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  158. Hatada, S., Nikkuni, K., Bentley, S. A., Kirby, S. & Smithies, O. Gene correction in hematopoietic progenitor cells by homologous recombination. Proc. Natl Acad. Sci. USA 97, 13807–13811 (2000).

    CAS  PubMed  Google Scholar 

  159. Wang, H. H. et al. Genome-scale promoter engineering by coselection MAGE. Nat. Methods 9, 591–593 (2012).

    PubMed  PubMed Central  Google Scholar 

  160. Liu, P.-Q. et al. Generation of a triple-gene knockout mammalian cell line using engineered zinc-finger nucleases. Biotechnol. Bioeng. 106, 97–105 (2010).

    CAS  PubMed  Google Scholar 

  161. Wang, J. et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat. Biotechnol. 33, 1256–1263 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  162. Hoban, M. D. et al. Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells. Blood 125, 2597–2604 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  163. Karakikes, I. et al. A comprehensive TALEN-based knockout library for generating human-induced pluripotent stem cell–based models for cardiovascular diseases. Circ. Res. 120, 1561–1571 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  164. Donoho, G., Jasin, M. & Berg, P. Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells. Mol. Cell. Biol. 18, 4070–4078 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  165. Lin, J. et al. Creating a monomeric endonuclease TALE-I-SceI with high specificity and low genotoxicity in human cells. Nucleic Acids Res. 43, 1112–1122 (2015).

    CAS  PubMed  Google Scholar 

  166. Cai, D., Cohen, K. B., Luo, T., Lichtman, J. W. & Sanes, J. R. Improved tools for the Brainbow toolbox. Nat. Methods 10, 540–547 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  167. Ding, Q. et al. Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell 12, 393–394 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by NIH grant RM1HG008525 “Center for Genomically Engineered Organs”.

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Correspondence to George M. Church.

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Competing interests

G.M.C. is a co-founder of Editas Medicine and eGenesis Bio and serves advisory roles in several companies involved in genome editing and engineering. A detailed listing of G.M.C.'s Tech Transfer, Advisory Roles and Funding Sources can be obtained from http://arep.med.harvard.edu/gmc/tech.html. R.C. declares no competing interests.

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Glossary

Homologous recombination

(HR). Exchange of sequence between two highly similar DNA molecules.

Homology-directed repair

(HDR). The process by which double-strand breaks (DSBs) in DNA are repaired when in the presence of an additional DNA moiety that has homology to the DNA sequence surrounding the DSB.

Protospacer adjacent motif

(PAM). A short, characteristic sequence (typically between 3 and 8 nucleotides in length) that the CRISPR-associated nuclease (such as Cas9 or Cpf1) recognizes before making its DNA break. Depending on the CRISPR system used, this sequence must either flank the 3′ end or 5′ end of the sequence of interest.

Reading

The use of massively parallel sequencing technologies to decipher nucleotide composition.

Writing

The use of either genome-editing tools and/or DNA synthesis to make desired changes in DNA sequence.

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Chari, R., Church, G. Beyond editing to writing large genomes. Nat Rev Genet 18, 749–760 (2017). https://doi.org/10.1038/nrg.2017.59

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