The emergence of sequence-specific nucleases that enable genome editing is revolutionizing basic and applied biology. Since the introduction of CRISPR–Cas9, genome editing has become widely used in transformable plants for characterizing gene function and improving traits, mainly by inducing mutations through non-homologous end joining of double-stranded breaks generated by CRISPR–Cas9. However, it would be highly desirable to perform precision gene editing in plants, especially in transformation-recalcitrant species. Recently developed Cas9 variants, novel RNA-guided nucleases and base-editing systems, and DNA-free CRISPR–Cas9 delivery methods now provide great opportunities for plant genome engineering. In this Review Article, we describe the current status of plant genome editing, focusing on newly developed genome editing tools and methods and their potential applications in plants. We also discuss the specific challenges facing plant genome editing, and future prospects.
Subscribe to Journal
Get full journal access for 1 year
only $5.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Bortesi, L. & Fischer, R. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol. Adv. 33, 41–52 (2015).
Brazelton, V. A. Jr et al. A quick guide to CRISPR sgRNA design tools. GM Crops Food 6, 266–276 (2015).
Lowe, K. et al. Morphogenic regulators Baby boom and Wuschel improve monocot transformation. Plant Cell 28, 1998–2015 (2016).
Zhang, Y. et al. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nat. Commun. 7, 12617 (2016).
Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).
Sprink, T., Eriksson, D., Schiemann, J. & Hartung, F. Regulatory hurdles for genome editing: process- vs. product-based approaches in different regulatory contexts. Plant Cell Rep. 35, 1493–1506 (2016).
Woo, J. W. et al. DNA-free genome editing in plants with preassembled CRISPR–Cas9 ribonucleoproteins. Nat. Biotechnol. 33, 1162–1164 (2015).
Svitashev, S. et al. Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nat. Commun. 7, 13274 (2016).
Liang, Z. et al. Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat. Commun. 8, 14261 (2017).
Latorre, A., Latorre, A. & Somoza, Á . Modified RNAs in CRISPR/Cas9: an old trick works again. Angew. Chem. Int. Ed. Engl. 55, 3548–3550 (2016).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).
Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).
Salvi, S., Druka, A., Milner, S. G. & Gruszka, D. in Biotechnological Approaches to Barley Improvement (eds Kumlehn, J. & Stein, N. ) 287–310 (Springer, 2014).
Zhang, D., Li, Z. & Li, J. F. Targeted gene manipulation in plants using the CRISPR/Cas technology. J. Genet. Genomics 43, 251–262 (2016).
Zhang, H. et al. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol. J. 12, 797–807 (2014).
Crosetto, N. et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat. Methods 10, 361–365 (2013).
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).
Frock, R. L. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. 33, 179–186 (2015).
Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR–Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015).
Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR–Cas9 for genome engineering. Cell 157, 1262–1278 (2014).
Schiml, S., Fauser, F. & Puchta, H. 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 (2014).
Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).
Kleinstiver, B. P. et al. High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).
Chylinski, K., Le Rhun, A. & Charpentier, E. The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biol. 10, 726–737 (2013).
Esvelt K. M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 10, 1116–1121 (2013).
Hou, Z. et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc. Natl Acad. Sci. USA 110, 15644–15649 (2013).
Kleinstiver, B. P. et al. Engineered CRISPR–Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).
Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).
Hu, X. et al. Expanding the range of CRISPR/Cas9 genome editing in rice. Mol. Plant 9, 943–945 (2016).
Karvelis, T. et al. Rapid characterization of CRISPR–Cas9 protospacer adjacent motif sequence elements. Genome Biol. 16, 253 (2015).
Steinert, J., Schiml, S., Fauser, F. & Puchta, H. Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus. Plant J. 84, 1295–1305 (2015).
Kaya, H. et al. Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9. Sci. Rep. 6, 26871 (2016).
Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).
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).
Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 31, 686–688 (2013).
Xu, R. et al. Generation of targeted mutant rice using a CRISPR-Cpf1 system. Plant Biotechnol. J. 15, 713–717 (2017).
Hu, X., Wang, C., Liu, Q., Fu, Y. & Wang, K. Targeted mutagenesis in rice using CRISPR-Cpf1 system. J. Genet. Genomics 44, 71–73 (2017).
Kim, H. et al. CRISPR/Cpf1-mediated DNA-free plant genome editing. Nat. Commun. 8, 14406 (2017).
Tang, X. et al. A CRISPR–Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat. Plants 3, 17018 (2017).
Zhang, H. Y. et al. MISSA 2.0: an updated synthetic biology toolbox for assembly of orthogonal CRISPR/Cas systems. Sci. Rep. 7, 41993 (2017).
Rouet, P., Smih, F. & Jasin, M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol. Cell. Biol. 14, 8096–8106 (1994).
Puchta, H., Dujon, B. & Hohn, B. 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 (1996).
Puchta, H. & Fauser, F. Synthetic nucleases for genome engineering in plants: prospects for a bright future. Plant J. 78, 727–741 (2014).
Voytas, D. F. & Gao, C. Precision genome engineering and agriculture: opportunities and regulatory challenges. PLoS Biol. 12, e1001877 (2014).
Svitashev, S. et al. Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiol. 169, 931–945 (2015).
Endo, M., Mikami, M. & Toki, S. Biallelic gene targeting in rice. Plant Physiol. 170, 667–677 (2016).
Baltes, N. J., Gil-Humanes, J., Cermak, T., Atkins, P. A. & Voytas D. F. DNA replicons for plant genome engineering. Plant Cell 26, 151–163 (2014).
Čermák, T., Baltes, N. J., Čegan, R., Zhang, Y. & Voytas, D. F. High-frequency, precise modification of the tomato genome. Genome Biol. 16, 232 (2015).
Butler, N. M., Baltes, N. J., Voytas, D. F. & Douches, D. S. Geminivirus-mediated genome editing in potato (Solanum tuberosum L.) using sequence-specific nucleases. Front. Plant Sci. 7, 1045 (2016).
Gil-Humanes, J. et al. High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9. Plant J. 89, 1251–1262 (2017).
Wang, M. et al. Gene targeting by homology-directed repair in rice using a geminivirus-based CRISPR/Cas9 System. Mol. Plant 10, 1007–1010 (2017).
Weinthal, D. M., Taylor, R. A. & Tzfira, T. Nonhomologous end joining-mediated gene replacement in plant cells. Plant Physiol. 162, 390–400 (2013).
Li, J. et al. Gene replacements and insertions in rice by intron targeting using CRISPR–Cas9. Nat. Plants 2, 16139 (2016).
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).
Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).
Ma, Y. et al. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods 13, 1029–1035 (2016).
Hess, G. et al. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat. Methods 13, 1036–1042 (2016).
Yang, L. et al. Engineering and optimising deaminase fusions for genome editing. Nat. Commun. 7, 13330 (2016).
Li, J., Sun, Y., Du, J., Zhao, Y. & Xia, L. Generation of targeted point mutations in rice by a modified CRISPR/Cas9 system. Mol. Plant 10, 526–529 (2017).
Lu, Y. & Zhu, J. K. Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 System. Mol. Plant 10, 523–525 (2017).
Zong, Y. et al. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat. Biotechnol. 35, 438–440 (2017).
Ren, B. et al. A CRISPR/Cas9 toolkit for efficient targeted base editing to induce genetic variations in rice. Sci. China Life Sci. 60, 516–519 (2017).
Shimatani, Z. et al. Targeted base editing in rice and tomato using a CRISPR–Cas9 cytidine deaminase fusion. Nat. Biotechnol. 35, 441–443 (2017).
Kim, Y. B. et al. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat. Biotechnol. 35, 371–376 (2017).
Carlson, J. C., Badran, A. H., Guggiana-Nilo, D. A. & Liu, D. R. Negative selection and stringency modulation in phage-assisted continuous evolution. Nat. Chem. Biol. 10, 216–222 (2014).
Piatek, A. et al. RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors. Plant Biotechnol. J. 13, 578–589 (2015).
Lowder, L. G. et al. A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. 169, 971–985 (2015).
Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).
Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).
Deng, W., Shi, X., Tjian, R., Lionnet, T. & Singer, R. H. CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells. Proc. Natl Acad. Sci. USA 112, 11870–11875 (2015).
Ma, H. et al. Multicolor CRISPR labeling of chromosomal loci in human cells. Proc. Natl Acad. Sci. USA 112, 3002–3007 (2015).
Ochiai, H., Sugawara, T. & Yamamoto, T. Simultaneous live imaging of the transcription and nuclear position of specific genes. Nucleic Acids Res. 43, e127 (2015).
Hilton, I. B. et al. Epigenome editing by a CRISPR–Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 33, 510–517 (2015).
Thakore, P. I. et al. Highly specific epigenome editing by CRISPR–Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12, 1143–1149 (2015).
Vojta, A. et al. Repurposing the CRISPR–Cas9 system for targeted DNA methylation. Nucleic Acids Res. 44, 5615–5628 (2016).
Tak, Y. G. & Farnham, P. J. Making sense of GWAS: using epigenomics and genome engineering to understand the functional relevance of SNPs in non-coding regions of the human genome. Epigenetics Chromatin 8, 57 (2015).
Sadhu, M. J., Bloom, J. S., Day, L. & Kruglyak, L. CRISPR-directed mitotic recombination enables genetic mapping without crosses. Science 352, 1113–1116 (2016).
Østerberg, J. T. et al. Accelerating the domestication of new crops: feasibility and approaches. Trends Plant Sci. 22, 373–384 (2017).
Ali, Z. et al. CRISPR/Cas9-mediated viral interference in plants. Genome Biol. 16, 238 (2015).
Baltes, N. J. et al. Conferring resistance to geminiviruses with the CRISPR–Cas prokaryotic immune system. Nat. Plants 1, 15145 (2015).
Ji, X. et al. Establishing a CRISPR–Cas-like immune system conferring DNA virus resistance in plants. Nat. Plants 1, 15144 (2015).
Miao, J. et al. Targeted mutagenesis in rice using CRISPR-Cas system. Cell Res. 23, 1233 (2013).
Xu, R. et al. Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice. Rice 7, 5 (2014).
Wang, Y. et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat. Biotechnol. 32, 947–951 (2014).
Ito, Y. et al. CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening. Biochem. Biophys. Res. Commun. 467, 76–82 (2015).
de Toledo Thomazella, D. P., Brail, Q., Dahlbeck, D. & Staskawicz, B. J. CRISPR–Cas9 mediated mutagenesis of a DMR6 ortholog in tomato confers broad-spectrum disease resistance. Preprint at http://biorxiv.org/content/early/2016/07/20/064824 (2016).
Wang, F. et al. Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PLoS ONE 11, e0154027 (2016).
Shi, J. et al. ARGOS8 variants generated by CRISPR–Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnol. J. 15, 207–216 (2016).
Sun, Y. et al. Generation of high-amylose rice through CRISPR/Cas9-mediated targeted mutagenesis of starch branching enzymes. Front. Plant Sci.https://doi.org/10.3389/fpls.2017.00298 (2017).
We apologize for the omission of any references that could not be included in this Review Article due to the space constraints. The work in the authors’ labs was supported by grants from the National Transgenic Science and Technology Program (2016ZX08010-002), the National Key Research and Development Program of China (2016YFD0100602), the Scientific Program of Beijing Municipal Commission of Science and Technology (Z171100001517001), the Chinese Academy of Sciences (XDB11030500), and the National Natural Science Foundation of China (31672015).
The authors declare no competing financial interests.
About this article
Cite this article
Yin, K., Gao, C. & Qiu, J. Progress and prospects in plant genome editing. Nature Plants 3, 17107 (2017). https://doi.org/10.1038/nplants.2017.107
Journal of Hazardous Materials (2021)
High-throughput phenotypic screening of random genomic fragments in transgenic rice identified novel drought tolerance genes
Theoretical and Applied Genetics (2020)
Conventional and Molecular Techniques from Simple Breeding to Speed Breeding in Crop Plants: Recent Advances and Future Outlook
International Journal of Molecular Sciences (2020)
Applications in Plant Sciences (2020)
Trends in Plant Science (2020)