Skip to main content
SpringerLink
Log in

Homology-based double-strand break-induced genome engineering in plants

  • Review
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

This review summarises the recent progress in DSB-induced gene targeting by homologous recombination in plants. We are getting closer to efficiently inserting genes or precisely exchanging single amino acids.

Abstract

Although the basic features of double-strand break (DSB)-induced genome engineering were established more than 20 years ago, only in recent years has the technique come into the focus of plant biologists. Today, most scientists apply the recently discovered CRISPR/Cas system for inducing site-specific DSBs in genes of interest to obtain mutations by non-homologous end joining (NHEJ), which is the prevailing and often imprecise mechanism of DSB repair in somatic plant cells. However, predefined changes like the site-specific insertion of foreign genes or an exchange of single amino acids can be achieved by DSB-induced homologous recombination (HR). Although DSB induction drastically enhances the efficiency of HR, the efficiency is still about two orders of magnitude lower than that of NHEJ. Therefore, significant effort have been put forth to improve DSB-induced HR based technologies. This review summarises the previous studies as well as discusses the most recent developments in using the CRISPR/Cas system to improve these processes for plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Baltes NJ, Gil-Humanes J, Cermak T, Atkins PA, Voytas DF (2014) DNA replicons for plant genome engineering. Plant Cell 26:151–163

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Beetham PR, Kipp PB, Sawycky XL, Arntzen CJ, May GD (1999) A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations. Proc Natl Acad Sci USA 96:8774–8778

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Čermák T, Baltes NJ, Čegan R, Zhang Y, Voytas DF (2015) High-frequency, precise modification of the tomato genome. Genome Biol 16:232

    Article  PubMed  PubMed Central  Google Scholar 

  • Charbonnel C, Allain E, Gallego ME, White CI (2011) Kinetic analysis of DNA double-strand break repair pathways in Arabidopsis. DNA Repair 10:611–619

    Article  CAS  PubMed  Google Scholar 

  • de Pater S, Pinas JE, Hooykaas PJJ, van der Zaal Bert J (2013) ZFN-mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium-mediated floral dip transformation. Plant Biotechnol J 11:510–515

    Article  CAS  PubMed  Google Scholar 

  • Doetschman T, Gregg RG, Maeda N, Hooper ML, Melton DW, Thompson S, Smithies O (1987) Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330:576–578

    Article  CAS  PubMed  Google Scholar 

  • Dong C, Beetham P, Vincent K, Sharp P (2006) Oligonucleotide-directed gene repair in wheat using a transient plasmid gene repair assay system. Plant Cell Rep 25:457–465

    Article  CAS  PubMed  Google Scholar 

  • Endo M, Ishikawa Y, Osakabe K, Nakayama S, Kaya H, Araki T, Shibahara K-I, Abe K, Ichikawa H, Valentine L, Hohn B, Toki S (2006) Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants. EMBO J 25:5579–5590

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Endo M, Mikami M, Toki S (2016) Biallelic Gene Targeting in Rice. Plant Physiol 170(2):667–677

    Article  PubMed  Google Scholar 

  • Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM (2013) Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods 10:1116–1121

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Even-Faitelson L, Samach A, Melamed-Bessudo C, Avivi-Ragolsky N, Levy AA (2011) Localized egg-cell expression of effector proteins for targeted modification of the Arabidopsis genome. Plant J 68:929–937

    Article  CAS  PubMed  Google Scholar 

  • Fauser F, Roth N, Pacher M, Ilg G, Sánchez-Fernández R, Biesgen C, Puchta H (2012) In planta gene targeting. Proc Natl Acad Sci USA 109:7535–7540

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fauser F, Schiml S, Puchta H (2014) Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 79:348–359

    Article  CAS  PubMed  Google Scholar 

  • Hartung F, Suer S, Puchta H (2007) Two closely related RecQ helicases have antagonistic roles in homologous recombination and DNA repair in Arabidopsis thaliana. Proc Natl Acad Sci USA 104:18836–18841

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 (New York, N.Y.) 337:816–821

    Article  CAS  Google Scholar 

  • Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales APW, Li Z, Peterson RT, Yeh J-RJ, Aryee MJ, Joung JK (2015) Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523:481–485

    Article  PubMed  PubMed Central  Google Scholar 

  • Knoll A, Puchta H (2011) The role of DNA helicases and their interaction partners in genome stability and meiotic recombination in plants. J Exp Bot 62:1565–1579

    Article  CAS  PubMed  Google Scholar 

  • Kwon Y-I, Abe K, Osakabe K, Endo M, Nishizawa-Yokoi A, Saika H, Shimada H, Toki S (2012) Overexpression of OsRecQl4 and/or OsExo1 enhances DSB-induced homologous recombination in rice. Plant Cell Physiol 53:2142–2152

    Article  CAS  PubMed  Google Scholar 

  • Li Z, Liu Z-B, Xing A, Moon BP, Koellhoffer JP, Huang L, Ward RT, Clifton E, Falco SC, Cigan AM (2015) Cas9-guide RNA directed genome editing in soybean. Plant Physiol 169:960–970

    Article  PubMed  PubMed Central  Google Scholar 

  • Ma H, Naseri A, Reyes-Gutierrez P, Wolfe SA, Zhang S, Pederson T (2015) Multicolor CRISPR labeling of chromosomal loci in human cells. Proc Natl Acad Sci USA 112:3002–3007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mahfouz MM, Li L, Shamimuzzaman M, Wibowo A, Fang X, Zhu J-K (2011) De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks. Proc Natl Acad Sci USA 108:2623–2628

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148

    Article  CAS  PubMed  Google Scholar 

  • Nassif N, Penney J, Pal S, Engels WR, Gloor GB (1994) Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair. Mol Cell Biol 14:1613–1625

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nishizawa-Yokoi A, Endo M, Osakabe K, Saika H, Toki S (2014) Precise marker excision system using an animal-derived piggyBac transposon in plants. Plant J 77:454–463

    Article  CAS  PubMed  Google Scholar 

  • Nishizawa-Yokoi A, Nonaka S, Osakabe K, Saika H, Toki S (2015) A universal positive–negative selection system for gene targeting in plants combining an antibiotic resistance gene and its antisense RNA. Plant Physiol 169:362–370

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Offringa R, de Groot MJ, Haagsman HJ, Does MP, van den Elzen PJ, Hooykaas PJ (1990) Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation. EMBO J 9:3077–3084

    CAS  PubMed  PubMed Central  Google Scholar 

  • Okuzaki A, Toriyama K (2004) Chimeric RNA/DNA oligonucleotide-directed gene targeting in rice. Plant Cell Rep 22:509–512

    Article  CAS  PubMed  Google Scholar 

  • Osman K, Higgins JD, Sanchez-Moran E, Armstrong SJ, Franklin FCH (2011) Pathways to meiotic recombination in Arabidopsis thaliana. New Phytol 190:523–544

    Article  CAS  PubMed  Google Scholar 

  • Paszkowski J, Baur M, Bogucki A, Potrykus I (1988) Gene targeting in plants. EMBO J 7:4021–4026

    CAS  PubMed  PubMed Central  Google Scholar 

  • Piatek A, Ali Z, Baazim H, Li L, Abulfaraj A, Al-Shareef S, Aouida M, Mahfouz MM (2015) RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol J 13:578–589

    Article  CAS  PubMed  Google Scholar 

  • Puchta H (1998) Repair of genomic double-strand breaks in somatic plant cells by one-sided invasion of homologous sequences. Plant J 13:331–339

    Article  CAS  Google Scholar 

  • Puchta H (2002) Gene replacement by homologous recombination in plants. Plant Mol Biol 48:173–182

    Article  CAS  PubMed  Google Scholar 

  • Puchta H (2005) The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution. J Exp Bot 56:1–14

    Article  CAS  PubMed  Google Scholar 

  • Puchta H (2016) Using CRISPR/Cas in three dimensions: towards synthetic plant genomes, transcriptomes and epigenomes. Plant J. doi:10.1111/tpj.13100

    Google Scholar 

  • Puchta H, Fauser F (2013) Gene targeting in plants: 25 years later. Int J Dev Biol 57:629–637

    Article  CAS  PubMed  Google Scholar 

  • Puchta H, Fauser F (2014) Synthetic nucleases for genome engineering in plants: prospects for a bright future. Plant J 78:727–741

    Article  CAS  PubMed  Google Scholar 

  • Puchta H, Dujon B, Hohn B (1993) 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Puchta H, Dujon B, Hohn B (1996) Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination. Proc Natl Acad Sci USA 93:5055–5060

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qi Y, Zhang Y, Zhang F, Baller JA, Cleland SC, Ryu Y, Starker CG, Voytas DF (2013) Increasing frequencies of site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways. Genome Res 23:547–554

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ran FA, Cong Le, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186–191

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Recker J, Knoll A, Puchta H (2014) The Arabidopsis thaliana homolog of the helicase RTEL1 plays multiple roles in preserving genome stability. Plant Cell 26:4889–4902

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reiss B, Schubert I, Köpchen K, Wendeler E, Schell J, Puchta H (2000) RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium. Proc Natl Acad Sci USA 97:3358–3363

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sauer NJ, Narváez-Vásquez J, Mozoruk J, Miller RB, Warburg ZJ, Woodward MJ, Mihiret YA, Lincoln TA, Segami RE, Sanders SL, Walker KA, Beetham PR, Schöpke CR, Gocal GF (2016) Oligonucleotide-mediated genome editing provides precision and function to engineered nucleases and antibiotics in plants. Plant physiol 170:1917–1928

    Article  PubMed  PubMed Central  Google Scholar 

  • Schaeffer SM, Nakata PA (2015) CRISPR/Cas9-mediated genome editing and gene replacement in plants: transitioning from lab to field. Plant Sci 240:130–142

    Article  CAS  PubMed  Google Scholar 

  • Schiml S, Puchta H (2016) Revolutionizing plant biology: multiple ways of genome engineering by CRISPR/Cas. Plant methods 12:8

    Article  PubMed  PubMed Central  Google Scholar 

  • Schiml S, Fauser F, Puchta H (2014) The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny. Plant J 80:1139–1150

    Article  CAS  PubMed  Google Scholar 

  • Shaked H, Melamed-Bessudo C, Levy AA (2005) High-frequency gene targeting in Arabidopsis plants expressing the yeast RAD54 gene. Proc Natl Acad Sci USA 102:12265–12269

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shan Q, Wang Y, Chen K, Liang Z, Li J, Zhang Y, Zhang K, Liu J, Voytas DF, Zheng X, Zhang Y, Gao C (2013) Rapid and efficient gene modification in rice and Brachypodium using TALENs. Mol Plant 6:1365–1368

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shimatani Z, Nishizawa-Yokoi A, Endo M, Toki S, Terada R (2014) Positive–negative-selection-mediated gene targeting in rice. Front Plant Sci 5:748

    PubMed  Google Scholar 

  • Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM, Rock JM, Wu Y-Y, Katibah GE, Zhifang G, McCaskill D, Simpson MA, Blakeslee B, Greenwalt SA, Butler HJ, Hinkley SJ, Zhang L, Rebar EJ, Gregory PD, Urnov FD (2009) Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459:437–441

    Article  CAS  PubMed  Google Scholar 

  • Siebert R, Puchta H (2002) Efficient repair of genomic double-strand breaks by homologous recombination between directly repeated sequences in the plant genome. Plant Cell 14:1121–1131

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Steinert J, Schiml S, Fauser F, Puchta H (2015) Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus. Plant J cell mol biol 84(6):1295–1305

    Article  CAS  Google Scholar 

  • Svitashev S, Young JK, Schwartz C, Gao H, Falco SC, Cigan AM (2015) Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiol 169:931–945

    Article  PubMed  PubMed Central  Google Scholar 

  • Szostak JW, Orr-Weaver TL, Rothstein RJ, Stahl FW (1983) The double-strand-break repair model for recombination. Cell 33:25–35

    Article  CAS  PubMed  Google Scholar 

  • Terada R, Urawa H, Inagaki Y, Tsugane K, Iida S (2002) Efficient gene targeting by homologous recombination in rice. Nat Biotechnol 20:1030–1034

    Article  CAS  PubMed  Google Scholar 

  • Thomas KR, Capecchi MR (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51:503–512

    Article  CAS  PubMed  Google Scholar 

  • Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, Joung JK, Voytas DF (2009) High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459:442–445

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Voytas DF (2013) Plant genome engineering with sequence-specific nucleases. Annu Rev Plant Biol 64:327–350

    Article  CAS  PubMed  Google Scholar 

  • Watanabe K, Pacher M, Dukowic S, Schubert V, Puchta H, Schubert I (2009) The structural maintenance of chromosomes 5/6 complex promotes sister chromatid alignment and homologous recombination after DNA damage in Arabidopsis thaliana. Plant Cell 21:2688–2699

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Weeks DP, Spalding MH, Yang B (2016) Use of designer nucleases for targeted gene and genome editing in plants. Plant biotechnol J 14(2):483–495

    Article  CAS  PubMed  Google Scholar 

  • Wright DA, Townsend JA, Winfrey RJ, Irwin PA, Rajagopal J, Lonosky PM, Hall BD, Jondle MD, Voytas DF (2005) High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J 44:693–705

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Zhang F, Li X, Baller JA, Qi Y, Starker CG, Bogdanove AJ, Voytas DF (2013) Transcription activator-like effector nucleases enable efficient plant genome engineering. Plant Physiol 161:20–27

    Article  CAS  PubMed  Google Scholar 

  • Zhu T, Peterson DJ, Tagliani L, St Clair G, Baszczynski CL, Bowen B (1999) Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides. Proc Natl Acad Sci USA 96:8768–8773

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu T, Mettenburg K, Peterson DJ, Tagliani L, Baszczynski CL (2000) Engineering herbicide-resistant maize using chimeric RNA/DNA oligonucleotides. Nat Biotechnol 18:555–558

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We apologise to all colleagues in this field for not being able to cite all of the rapidly growing number of reports published on genome engineering in plants due to space limitations. Work on DSB-induced genome engineering in our lab has been funded by the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung (BMBF), the EU and the ERC (Advanced Grant COMREC).

Author information

Authors and Affiliations

  1. Botanical Institute II, Karlsruhe Institute of Technology, POB 6980, 76049, Karlsruhe, Germany

    Jeannette Steinert, Simon Schiml & Holger Puchta

Authors
  1. Jeannette Steinert
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Simon Schiml
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Holger Puchta
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Holger Puchta.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Communicated by T. Cardi.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Steinert, J., Schiml, S. & Puchta, H. Homology-based double-strand break-induced genome engineering in plants. Plant Cell Rep 35, 1429–1438 (2016). https://doi.org/10.1007/s00299-016-1981-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-016-1981-3

Keywords

Search

Navigation