Abstract
Key message
We summarize recent progress of CRISPR/Cas9-mediated gene targeting in plants, provide recommendations for designing gene-targeting vectors and highlight the potential of new technologies applicable to plants.
Abstract
Gene targeting (GT) is a tool of urgent need for plant biotechnology and breeding. It is based on homologous recombination that is able to precisely introduce desired modifications within a target locus. However, its low efficiency in higher plants is a major barrier for its application. Using site-specific nucleases, such as the recent CRISPR/Cas system, GT has become applicable in plants, via the induction of double-strand breaks, although still at a too low efficiency for most practical applications in crops. Recently, a variety of promising new improvements regarding the efficiency of GT has been reported by several groups. It turns out that GT can be enhanced by cell-type-specific expression of Cas nucleases, by the use of self-amplified GT-vector DNA or by manipulation of DNA repair pathways. Here, we highlight the most recent progress of GT in plants. Moreover, we provide suggestions on how to use the technology efficiently, based on the mechanisms of DNA repair, and highlight several of the newest GT strategies in yeast or mammals that are potentially applicable to plants. Using the full potential of GT technology will definitely help us pave the way in enhancing crop yields and food safety for an ecologically friendly agriculture.
Similar content being viewed by others
References
Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F (2017) RNA targeting with CRISPR-Cas13. Nature 550:280–284. https://doi.org/10.1038/nature24049
Adikusuma F, Piltz S, Corbett MA, Turvey M, McColl SR, Helbig KJ, Beard MR, Hughes J, Pomerantz RT, Thomas PQ (2018) Large deletions induced by Cas9 cleavage. Nature 560:E8–E9. https://doi.org/10.1038/s41586-018-0380-z
Aird EJ, Lovendahl KN, St. Martin A, Harris RS, Gordon WR (2018) Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. Commun Biol 1:816. https://doi.org/10.1038/s42003-018-0054-2
Baltes NJ, Gil-Humanes J, Cermak T, Atkins PA, Voytas DF (2014) DNA replicons for plant genome engineering. Plant Cell 26:151–163. https://doi.org/10.1105/tpc.113.119792
Begemann MB, Gray BN, January E, Gordon GC, He Y, Liu H, Wu X, Brutnell TP, Mockler TC, Oufattole M (2017) Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases. Sci Rep 7:11606. https://doi.org/10.1038/s41598-017-11760-6
Bent AF (2000) Arabidopsis in planta transformation. uses, mechanisms, and prospects for transformation of other species. Plant Physiol 124:1540–1547. https://doi.org/10.1104/pp.124.4.1540
Bothmer A, Phadke T, Barrera LA, Margulies CM, Lee CS, Buquicchio F, Moss S, Abdulkerim HS, Selleck W, Jayaram H, Myer VE, Cotta-Ramusino C (2017) Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus. Nat Commun 8:13905. https://doi.org/10.1038/ncomms13905
Caridi CP, D’Agostino C, Ryu T, Zapotoczny G, Delabaere L, Li X, Khodaverdian VY, Amaral N, Lin E, Rau AR, Chiolo I (2018) Nuclear F-actin and myosins drive relocalization of heterochromatic breaks. Nature 559:54–60. https://doi.org/10.1038/s41586-018-0242-8
Cermak T, Baltes NJ, Cegan R, Zhang Y, Voytas DF (2015) High-frequency, precise modification of the tomato genome. Genome Biol 16:232. https://doi.org/10.1186/s13059-015-0796-9
Cermak T, Curtin SJ, Gil-Humanes J, Cegan R, Kono TJY, Konecna E, Belanto JJ, Starker CG, Mathre JW, Greenstein RL, Voytas DF (2017) A multipurpose toolkit to enable advanced genome engineering in plants. Plant Cell 29:1196–1217. https://doi.org/10.1105/tpc.16.00922
Charpentier M, Khedher AHY, Menoret S, Brion A, Lamribet K, Dardillac E, Boix C, Perrouault L, Tesson L, Geny S, Cian A de, Itier JM, Anegon I, Lopez B, Giovannangeli C, Concordet JP (2018) CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair. Nat Commun 9:1133. https://doi.org/10.1038/s41467-018-03475-7
Dahan-Meir T, Filler-Hayut S, Melamed-Bessudo C, Bocobza S, Czosnek H, Aharoni A, Levy AA (2018) Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR/Cas9 system. Plant J 95:5–16. https://doi.org/10.1111/tpj.13932
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. https://doi.org/10.1038/sj.emboj.7601434
Endo M, Mikami M, Toki S (2016) Biallelic gene targeting in rice. Plant Physiol 170:667–677. https://doi.org/10.1104/pp.15.01663
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. https://doi.org/10.1111/j.1365-313X.2011.04741.x
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. https://doi.org/10.1073/pnas.1202191109
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. https://doi.org/10.1111/tpj.12554
Ferenczi A, Pyott DE, Xipnitou A, Molnar A (2017) Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA. Proc Natl Acad Sci USA 114:13567–13572. https://doi.org/10.1073/pnas.1710597114
Ferreira MG, Cooper JP (2004) Two modes of DNA double-strand break repair are reciprocally regulated through the fission yeast cell cycle. Genes Dev 18:2249–2254. https://doi.org/10.1101/gad.315804
Ge X, Wang H, Cao K (2008) Transformation by T-DNA integration causes highly sterile phenotype independent of transgenes in Arabidopsis thaliana. Plant Cell Rep 27:1341–1348. https://doi.org/10.1007/s00299-008-0561-6
Gil-Humanes J, Wang Y, Liang Z, Shan Q, Ozuna CV, Sanchez-Leon S, Baltes NJ, Starker C, Barro F, Gao C, Voytas DF (2017) High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9. Plant J 89:1251–1262. https://doi.org/10.1111/tpj.13446
Gu B, Posfai E, Rossant J (2018) Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat Biotechnol 36:632–637. https://doi.org/10.1038/nbt.4166
Hahn F, Eisenhut M, Mantegazza O, Weber APM (2018) Homology-directed repair of a defective glabrous gene in Arabidopsis with Cas9-based gene targeting. Front Plant Sci 9:424. https://doi.org/10.3389/fpls.2018.00424
Hanin M, Volrath S, Bogucki A, Briker M, Ward E, Paszkowski J (2001) Gene targeting in Arabidopsis. Plant J 28:671–677. https://doi.org/10.1046/j.1365-313x.2001.01183.x
Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31:827–832. https://doi.org/10.1038/nbt.2647
Kaya H, Mikami M, Endo A, Endo M, Toki S (2016) Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9. Sci Rep 6:26871. https://doi.org/10.1038/srep26871
Kim D, Lim K, Kim S-T, Yoon S-H, Kim K, Ryu S-M, Kim J-S (2017) Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat Biotechnol 35:475–480. https://doi.org/10.1038/nbt.3852
Kirik A, Salomon S, Puchta H (2000) Species-specific double-strand break repair and genome evolution in plants. EMBO J 19:5562–5566. https://doi.org/10.1093/emboj/19.20.5562
Kirik A, Pecinka A, Wendeler E, Reiss B (2006) The chromatin assembly factor subunit FASCIATA1 is involved in homologous recombination in plants. Plant Cell 18:2431–2442. https://doi.org/10.1105/tpc.106.045088
La Russa MF, Qi LS (2015) The new state of the art: Cas9 for gene activation and repression. Mol Cell Biol 35:3800–3809. https://doi.org/10.1128/MCB.00512-15
Li J, Sun Y, Du J, Zhao Y, Xia L (2017) Generation of targeted point mutations in rice by a modified CRISPR/Cas9 system. Mol Plant 10:526–529. https://doi.org/10.1016/j.molp.2016.12.001
Li J, Zhang X, Sun Y, Zhang J, Du W, Guo X, Li S, Zhao Y, Xia L (2018a) Efficient allelic replacement in rice by gene editing: a case study of the NRT1.1B gene. J Integr Plant Biol 60:536–540. https://doi.org/10.1111/jipb.12650
Li C, Zong Y, Wang Y, Jin S, Zhang D, Song Q, Zhang R, Gao C (2018b) Expanded base editing in rice and wheat using a Cas9-adenosine deaminase fusion. Genome Biol 19:59. https://doi.org/10.1186/s13059-018-1443-z
Li S, Li J, Zhang J, Du W, Fu J, Sutar S, Zhao Y, Xia L (2018c) Synthesis-dependent repair of Cpf1-induced double strand DNA breaks enables targeted gene replacement in rice. J Exp Bot 69:4715–4721. https://doi.org/10.1093/jxb/ery245
Ma M, Zhuang F, Hu X, Wang B, Wen X-Z, Ji J-F, Xi JJ (2017) Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-avidin/biotin-donor DNA system. Cell Res 27:578–581. https://doi.org/10.1038/cr.2017.29
Mao Z, Bozzella M, Seluanov A, Gorbunova V (2008) DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells. Cell Cycle 7:2902–2906. https://doi.org/10.4161/cc.7.18.6679
Mao Y, Zhang Z, Feng Z, Wei P, Zhang H, Botella JR, Zhu J-K (2016) Development of germ-line-specific CRISPR–Cas9 systems to improve the production of heritable gene modifications in Arabidopsis. Plant Biotechnol J 14:519–532. https://doi.org/10.1111/pbi.12468
Miki D, Zhang W, Zeng W, Feng Z, Zhu J-K (2018) CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation. Nat Commun 9:1967. https://doi.org/10.1038/s41467-018-04416-0
Miura H, Quadros RM, Gurumurthy CB, Ohtsuka M (2018) Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat Protoc 13:195–215. https://doi.org/10.1038/nprot.2017.153
Orel N, Kyryk A, Puchta H (2003) Different pathways of homologous recombination are used for the repair of double-strand breaks within tandemly arranged sequences in the plant genome. Plant J 35:604–612. https://doi.org/10.1046/j.1365-313X.2003.01832.x
Orozco BM, Kong L-J, Batts LA, Elledge S, Hanley-Bowdoin L (2000) The multifunctional character of a geminivirus replication protein is reflected by its complex oligomerization properties. J Biol Chem 275:6114–6122. https://doi.org/10.1074/jbc.275.9.6114
Paszkowski J, Baur M, Bogucki A, Potrykus I (1988) Gene targeting in plants. EMBO J 7:4021–4026
Pater S de, Klemann B, Hooykaas PJJ (2018) True gene-targeting events by CRISPR/Cas-induced DSB repair of the PPO locus with an ectopically integrated repair template. Sci Rep 8:3338. https://doi.org/10.1038/s41598-018-21697-z
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. https://doi.org/10.1046/j.1365-313X.1998.00035.x
Puchta H (2005) The repair of double-strand breaks in plants: mechanisms and consequences for genome evolution. J Exp Bot 56:1–14. https://doi.org/10.1093/jxb/eri025
Puchta H (2017) Applying CRISPR/Cas for genome engineering in plants: the best is yet to come. Curr Opin Plant Biol 36:1–8. https://doi.org/10.1016/j.pbi.2016.11.011
Puchta H, Fauser F (2013) Gene targeting in plants: 25 years later. Int J Dev Biol 57:629–637. https://doi.org/10.1387/ijdb.130194hp
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. https://doi.org/10.1073/pnas.93.10.5055
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. https://doi.org/10.1101/gr.145557.112
Quadros RM, Miura H, Harms DW, Akatsuka H, Sato T, Aida T, Redder R, Richardson GP, Inagaki Y, Sakai D, Buckley SM, Seshacharyulu P, Batra SK, Behlke MA, Zeiner SA, Jacobi AM, Izu Y, Thoreson WB, Urness LD, Mansour SL, Ohtsuka M, Gurumurthy CB (2017) 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. https://doi.org/10.1186/s13059-017-1220-4
Reiss B, Klemm M, Kosak H, Schell J (1996) RecA protein stimulates homologous recombination in plants. Proc Natl Acad Sci USA 93:3094–3098. https://doi.org/10.1073/pnas.93.7.3094
Reiss B, Schubert I, Kopchen 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. https://doi.org/10.1073/pnas.97.7.3358
Rolloos M, Hooykaas PJJ, van der Zaal BJ (2015) Enhanced targeted integration mediated by translocated I-SceI during the Agrobacterium mediated transformation of yeast. Sci Rep 5:8345. https://doi.org/10.1038/srep08345
Roy KR, Smith JD, Vonesch SC, Lin G, Tu CS, Lederer AR, Chu A, Suresh S, Nguyen M, Horecka J, Tripathi A, Burnett WT, Morgan MA, Schulz J, Orsley KM, Wei W, Aiyar RS, Davis RW, Bankaitis VA, Haber JE, Salit ML, St Onge RP, Steinmetz LM (2018) Multiplexed precision genome editing with trackable genomic barcodes in yeast. Nat Biotechnol 36:512–520. https://doi.org/10.1038/nbt.4137
Saito S, Maeda R, Adachi N (2017) Dual loss of human POLQ and LIG4 abolishes random integration. Nat Commun 8:16112. https://doi.org/10.1038/ncomms16112
Salomon S, Puchta H (1998) Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. EMBO J 17:6086–6095. https://doi.org/10.1093/emboj/17.20.6086
Savic N, Ringnalda FC, Lindsay H, Berk C, Bargsten K, Li Y, Neri D, Robinson MD, Ciaudo C, Hall J, Jinek M, Schwank G (2018) Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair. eLife 7:e33761. https://doi.org/10.7554/eLife.33761
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. https://doi.org/10.1111/tpj.12704
Schindele P, Wolter F, Puchta H (2018) Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13. FEBS Lett 592:1954–1967. https://doi.org/10.1002/1873-3468.13073
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. https://doi.org/10.1073/pnas.0502601102
Sprunck S, Rademacher S, Vogler F, Gheyselinck J, Grossniklaus U, Dresselhaus T (2012) Egg cell-secreted EC1 triggers sperm cell activation during double fertilization. Science 338:1093–1097. https://doi.org/10.1126/science.1223944
Steffen JG, Kang I-H, Macfarlane J, Drews GN (2007) Identification of genes expressed in the Arabidopsis female gametophyte. Plant J 51:281–292. https://doi.org/10.1111/j.1365-313X.2007.03137.x
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 84:1295–1305. https://doi.org/10.1111/tpj.13078
Steinert J, Schiml S, Puchta H (2016) Homology-based double-strand break-induced genome engineering in plants. Plant Cell Rep 35:1429–1438. https://doi.org/10.1007/s00299-016-1981-3
Sun Y, Zhang X, Wu C, He Y, Ma Y, Hou H, Guo X, Du W, Zhao Y, Xia L (2016a) Engineering herbicide-resistant rice plants through CRISPR/Cas9-mediated homologous recombination of acetolactate synthase. Mol Plant 9:628–631. https://doi.org/10.1016/j.molp.2016.01.001
Sun Y, Li J, Xia L (2016b) Precise genome modification via sequence-specific nucleases-mediated gene targeting for crop improvement. Front Plant Sci 7:1928. https://doi.org/10.3389/fpls.2016.01928
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. https://doi.org/10.1104/pp.15.00793
Svitashev S, Schwartz C, Lenderts B, Young JK, Mark Cigan A (2016) Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nat Commun 7:13274. https://doi.org/10.1038/ncomms13274
Tsakraklides V, Brevnova E, Stephanopoulos G, Shaw AJ (2015) Improved gene targeting through cell cycle synchronization. PLoS One 10:e0133434. https://doi.org/10.1371/journal.pone.0133434
Twell D, Yamaguchi J, McCormick S (1990) Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis. Development 109:705–713
van Kregten M, Pater S de, Romeijn R, van Schendel R, Hooykaas PJJ, Tijsterman M (2016) T-DNA integration in plants results from polymerase-θ-mediated DNA repair. Nat Plants 2:16164. https://doi.org/10.1038/nplants.2016.164
Wang Z-P, Xing H-L, Dong L, Zhang H-Y, Han C-Y, Wang X-C, Chen Q-J (2015) Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biol 16:144. https://doi.org/10.1186/s13059-015-0715-0
Wang M, Lu Y, Botella JR, Mao Y, Hua K, Zhu J-K (2017) Gene targeting by homology-directed repair in rice using a geminivirus-based CRISPR/Cas9 system. Mol Plant 10:1007–1010. https://doi.org/10.1016/j.molp.2017.03.002
Wolter F, Puchta H (2018) The CRISPR/Cas revolution reaches the RNA world: Cas13, a new Swiss army knife for plant biologists. Plant J 94:767–775. https://doi.org/10.1111/tpj.13899
Wolter F, Klemm J, Puchta H (2018) Efficient in planta gene targeting in Arabidopsis using egg cell-specific expression of the Cas9 nuclease of Staphylococcus aureus. Plant J 94:735–746. https://doi.org/10.1111/tpj.13893
Yan L, Wei S, Wu Y, Hu R, Li H, Yang W, Xie Q (2015) High-efficiency genome editing in Arabidopsis using YAO promoter-driven CRISPR/Cas9 system. Mol Plant 8:1820–1823. https://doi.org/10.1016/j.molp.2015.10.004
Yang D, Scavuzzo MA, Chmielowiec J, Sharp R, Bajic A, Borowiak M (2016) Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases. Sci Rep 6:21264. https://doi.org/10.1038/srep21264
Yourik P, Fuchs RT, Mabuchi M, Curcuru JL, Robb GB (2019) Staphylococcus aureus Cas9 is a multiple-turnover enzyme. RNA 25:35–44. https://doi.org/10.1261/rna.067355.118
Zelensky AN, Schimmel J, Kool H, Kanaar R, Tijsterman M (2017) Inactivation of Pol θ and C-NHEJ eliminates off-target integration of exogenous DNA. Nat Commun 8:66. https://doi.org/10.1038/s41467-017-00124-3
Zong Y, Wang Y, Li C, Zhang R, Chen K, Ran Y, Qiu J-L, Wang D, Gao C (2017) Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat Biotechnol 35:438–440. https://doi.org/10.1038/nbt.3811
Acknowledgements
We apologize to all colleagues in this field, as due to space limitations, we were not able to cite all relevant reports on the rapidly growing aspects of genome engineering. We are thankful for the funding support from the Ministry of Science and Technology of Taiwan, ROC (MOST 106-2917-I-564-007-A1) and the Bundesministerium für Forschung und Technologie (100334243 SophGenTom). We also acknowledge Amy Whitbread for English editing.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
All authors declare that they have no conflict of interest.
Additional information
Communicated by Laurence Tomlinson.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Huang, TK., Puchta, H. CRISPR/Cas-mediated gene targeting in plants: finally a turn for the better for homologous recombination. Plant Cell Rep 38, 443–453 (2019). https://doi.org/10.1007/s00299-019-02379-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-019-02379-0