Abstract
Crop improvement by genome editing involves the targeted alteration of genes to improve plant traits, such as stress tolerance, disease resistance or nutritional content. Techniques for the targeted modification of genomes have evolved from generating random mutations to precise base substitutions, followed by insertions, substitutions and deletions of small DNA fragments, and are finally starting to achieve precision manipulation of large DNA segments. Recent developments in base editing, prime editing and other CRISPR-associated systems have laid a solid technological foundation to enable plant basic research and precise molecular breeding. In this Review, we systematically outline the technological principles underlying precise and targeted genome-modification methods. We also review methods for the delivery of genome-editing reagents in plants and outline emerging crop-breeding strategies based on targeted genome modification. Finally, we consider potential future developments in precise genome-editing technologies, delivery methods and crop-breeding approaches, as well as regulatory policies for genome-editing products.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Tilman, D., Balzer, C., Hill, J. & Befort, B. L. Global food demand and the sustainable intensification of agriculture. Proc. Natl Acad. Sci. USA 108, 20260–20264 (2011).
Meyer, R. S. & Purugganan, M. D. Evolution of crop species: genetics of domestication and diversification. Nat. Rev. Genet. 14, 840–852 (2013).
Holme, I. B., Gregersen, P. L. & Brinch-Pedersen, H. Induced genetic variation in crop plants by random or targeted mutagenesis: convergence and differences. Front. Plant. Sci. 10, 1468 (2019).
Chen, K., Wang, Y., Zhang, R., Zhang, H. & Gao, C. CRISPR/Cas genome eediting and precision plant breeding in agriculture. Annu. Rev. Plant. Biol. 70, 667–697 (2019).
Jinek, M. et al. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Gaj, T., Gersbach, C. A. & Barbas, C. F. III ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397–405 (2013).
Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).
Liu, G., Lin, Q., Jin, S. & Gao, C. The CRISPR-Cas toolbox and gene editing technologies. Mol. Cell 82, 333–347 (2022).
Liu, Z. H. et al. Precise editing of methylated cytosine in Arabidopsis thaliana using a human APOBEC3Bctd–Cas9 fusion. Sci. China Life Sci. 65, 219–222 (2022).
Tang, S. et al. Targeted DNA demethylation produces heritable epialleles in rice. Sci. China Life Sci. 65, 753–756 (2022).
Gao, C. Genome engineering for crop improvement and future agriculture. Cell 184, 1621–1635 (2021).
Rao, Y., Yang, X., Pan, C., Wang, C. & Wang, K. Advance of clustered regularly interspaced short palindromic repeats–Cas9 system and its application in crop improvement. Front. Plant. Sci. 13, 839001 (2022).
Chen, Z., Debernardi, J. M., Dubcovsky, J. & Gallavotti, A. Recent advances in crop transformation technologies. Nat. Plants 8, 1343–1351 (2022).
Altpeter, F. et al. Advancing crop transformation in the era of genome editing. Plant. Cell 28, 1510–1520 (2016).
Fauser, F., Schiml, S. & Puchta, H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant. J. 79, 348–359 (2014).
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).
Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).
Alanis-Lobato, G. et al. Frequent loss of heterozygosity in CRISPR–Cas9-edited early human embryos. Proc. Natl Acad. Sci. USA 118, e2004832117 (2021).
Shan, Q. et al. Targeted genome modification of crop plants using a CRISPR–Cas system. Nat. Biotechnol. 31, 686–688 (2013). This study marks the first application of CRISPR–Cas9 technology in plants, generating mutants with the desired edits.
Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).
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). This work combined deaminase with CRISPR for the first time to achieve precise cytosine editing without double-strand breaks at the single-base level.
Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, 6305 (2016).
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).
Zong, Y. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat. Biotechnol. 36, 950–953 (2018).
Jin, S. et al. Rationally designed APOBEC3B cytosine base editors with improved specificity. Mol. Cell 79, 728–740 e726 (2020).
Liang, Y., Chen, F., Wang, K. & Lai, L. Base editors: development and applications in biomedicine. Front. Med. 17, 359–387 (2023).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Huang, J. et al. Discovery of deaminase functions by structure-based protein clustering. Cell 186, 3182–3195.e3114 (2023). This work used artificial-intelligence-assisted structural clustering methods for mining novel enzymes suitable for genome editing in eukaryote.
Cortizas, E. M. et al. UNG protects B cells from AID-induced telomere loss. J. Exp. Med. 213, 2459–2472 (2016).
Kurt, I. C. et al. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. 39, 41–46 (2021).
Zhao, D. et al. Glycosylase base editors enable C-to-A and C-to-G base changes. Nat. Biotechnol. 39, 35–40 (2021).
Sretenovic, S. et al. Exploring C-To-G base editing in rice, tomato, and poplar. Front. Genome Edit. 3, 756766 (2021).
Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017). This work created the first adenine base editor through extensive directed evolution and protein engineering.
Li, C. et al. Expanded base editing in rice and wheat using a Cas9–adenosine deaminase fusion. Genome Biol. 19, 59 (2018).
Kang, B. C. et al. Precision genome engineering through adenine base editing in plants. Nat. Plants 4, 427–431 (2018).
Richter, M. F. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 38, 883–891 (2020).
Wei, C. et al. Efficient generation of homozygous substitutions in rice in one generation utilizing an rABE8e base editor. J. Integr. Plant. Biol. 63, 1595–1599 (2021).
Tong, H. et al. Programmable A-to-Y base editing by fusing an adenine base editor with an N-methylpurine DNA glycosylase. Nat. Biotechnol. 41, 1080–1084 (2023).
Chen, L. et al. Adenine transversion editors enable precise, efficient A*T-to-C*G base editing in mammalian cells and embryos. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01821-9 (2023).
Wu, X. et al. Adenine base editor incorporating the N-methylpurine DNA glycosylase MPGv3 enables efficient A-to-K base editing in rice. Plant. Commun. 4, 100668 (2023).
Tong, H. et al. Programmable deaminase-free base editors for G-to-Y conversion by engineered glycosylase. Natl Sci. Rev. 10, nwad143 (2023).
He, Y. et al. Protein language models-assisted optimization of a uracil-N-glycosylase variant enables programmable T-to-G and T-to-C base editing. Mol. Cell 84, 1257–1270.e6 (2024).
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019). This work described the development of prime editing and precisely achieved all types of base substitutions and small insertions or deletions without double-strand breaks in eukaryotes for the first time.
Zong, Y. et al. An engineered prime editor with enhanced editing efficiency in plants. Nat. Biotechnol. 40, 1394–1402 (2022). This work greatly increased the efficiency of prime editing in plants through protein engineering.
Ni, P. et al. Efficient and versatile multiplex prime editing in hexaploid wheat. Genome Biol. 24, 156 (2023).
Doman, J. L. et al. Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell 186, 3983–4002.e3926 (2023).
Ponnienselvan, K. et al. Addressing the dNTP bottleneck restricting prime editing activity. Preprint at bioRxiv https://doi.org/10.1101/2023.10.21.563443 (2023).
Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652.e5629 (2021).
Liu, P. et al. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nat. Commun. 12, 2121 (2021).
Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 40, 402–410 (2022).
Zhang, G. et al. Enhancement of prime editing via xrRNA motif-joined pegRNA. Nat. Commun. 13, 1856 (2022).
Li, X. et al. Enhancing prime editing efficiency by modified pegRNA with RNA G-quadruplexes. J. Mol. Cell Biol. 14, mjac022 (2022).
Li, X. et al. Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure. Nat. Commun. 13, 1669 (2022).
Xu, W. et al. A design optimized prime editor with expanded scope and capability in plants. Nat. Plants 8, 45–52 (2022).
Yu, G. et al. Prediction of efficiencies for diverse prime editing systems in multiple cell types. Cell 186, 2256–2272.e2223 (2023).
Jiang, Y. et al. Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP–IVS mutation in EPSPS. Mol. Plant. 15, 1646–1649 (2022).
Liu, B. et al. Targeted genome editing with a DNA-dependent DNA polymerase and exogenous DNA-containing templates. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01947-w (2023).
da Silva, J. F. et al. Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases. Preprint at bioRxiv https://doi.org/10.1101/2023.09.12.557440 (2023).
Wang, S. et al. Precise, predictable multi-nucleotide deletions in rice and wheat using APOBEC–Cas9. Nat. Biotechnol. 38, 1460–1465 (2020).
Dong, O. X. et al. Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR–Cas9. Nat. Commun. 11, 1178 (2020).
Gisler, B., Salomon, S. & Puchta, H. The role of double‐strand-break‐induced allelic homologous recombination in somatic plant cells. Plant. J. 32, 277–284 (2002).
Rönspies, M. et al. Massive crossover suppression by CRISPR–Cas-mediated plant chromosome engineering. Nat. Plants 8, 1153–1159 (2022).
Beying, N., Schmidt, C., Pacher, M., Houben, A. & Puchta, H. CRISPR–Cas9-mediated induction of heritable chromosomal translocations in Arabidopsis. Nat. Plants 6, 638–645 (2020).
Wang, J. et al. Efficient targeted insertion of large DNA fragments without DNA donors. Nat. Methods 19, 331–340 (2022).
Sun, C. et al. Precise integration of large DNA sequences in plant genomes using PrimeRoot editors. Nat. Biotechnol. 42, 316–327 (2023). This work achieved double-strand-break-independent precise targeted insertion of large DNA segments in plants.
Choi, J. et al. Precise genomic deletions using paired prime editing. Nat. Biotechnol. 40, 218–226 (2022).
Jiang, T., Zhang, X. O., Weng, Z. & Xue, W. Deletion and replacement of long genomic sequences using prime editing. Nat. Biotechnol. 40, 227–234 (2022).
Tansirichaiya, S., Rahman, M. A. & Roberts, A. P. The transposon registry. Mob. DNA 10, 40 (2019).
O’Donnell, K. A. Advances in functional genetic screening with transposons and CRISPR/Cas9 to illuminate cancer biology. Curr. Opin. Genet. Dev. 49, 85–94 (2018).
Liu, P. et al. CRISPR-targeted transposable element insertion for efficient plant genome engineering. Preprint at Research Square https://doi.org/10.21203/rs.3.rs-2679086/v1 (2023).
Pallares-Masmitja, M. et al. Find and cut-and-transfer (FiCAT) mammalian genome engineering. Nat. Commun. 12, 7071 (2021).
Klompe, S. E., Vo, P. L. H., Halpin-Healy, T. S. & Sternberg, S. H. Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration. Nature 571, 219–225 (2019).
Strecker, J. et al. RNA-guided DNA insertion with CRISPR-associated transposases. Science 365, 48–53 (2019).
Tou, C. J., Orr, B. & Kleinstiver, B. P. Precise cut-and-paste DNA insertion using engineered type V-K CRISPR-associated transposases. Nat. Biotechnol. 41, 968–979 (2023).
Lampe, G. D. et al. Targeted DNA integration in human cells without double-strand breaks using CRISPR-associated transposases. Nat. Biotechnol. 42, 87–98 (2023).
Wilkinson, M. E., Frangieh, C. J., Macrae, R. K. & Zhang, F. Structure of the R2 non-LTR retrotransposon initiating target-primed reverse transcription. Science 380, 301–308 (2023).
Hirano, N., Muroi, T., Takahashi, H. & Haruki, M. Site-specific recombinases as tools for heterologous gene integration. Appl. Microbiol. Biotechnol. 92, 227–239 (2011).
Anzalone, A. V. et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat. Biotechnol. 40, 731–740 (2022).
Yarnall, M. T. N. et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat. Biotechnol. 41, 500–512 (2023).
Wang, C. et al. dCas9-based gene editing for cleavage-free genomic knock-in of long sequences. Nat. Cell Biol. 24, 268–278 (2022).
Nasti, R. A. & Voytas, D. F. Attaining the promise of plant gene editing at scale. Proc. Natl Acad. Sci. USA 118, e2004846117 (2021).
Ghogare, R., Ludwig, Y., Bueno, G. M., Slamet-Loedin, I. H. & Dhingra, A. Genome editing reagent delivery in plants. Transgen. Res. 30, 321–335 (2021).
Svitashev, S., Schwartz, C., Lenderts, B., Young, J. K. & Mark Cigan, A. Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nat. Commun. 7, 13274 (2016). This work provides a method of efficient transient genome editing using ribonucleoprotein complexes through particle bombardment.
Liang, Z. et al. Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat. Commun. 8, 14261 (2017).
Liang, Z. et al. Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins. Nat. Protoc. 13, 413–430 (2018).
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).
Qiu, F. et al. Transient expression of a TaGRF4–TaGIF1 complex stimulates wheat regeneration and improves genome editing. Sci. China Life Sci. 65, 731–738 (2022).
Lowe, K. et al. Morphogenic regulators Baby boom and Wuschel improve monocot transformation. Plant. Cell 28, 1998–2015 (2016).
Liu, J. et al. Genome-scale sequence disruption following biolistic transformation in rice and maize. Plant. Cell 31, 368–383 (2019).
Pan, C. et al. Boosting plant genome editing with a versatile CRISPR–Combo system. Nat. Plants 8, 513–525 (2022).
Debernardi, J. M. et al. A GRF–GIF chimeric protein improves the regeneration efficiency of transgenic plants. Nat. Biotechnol. 38, 1274–1279 (2020).
Maher, M. F. et al. Plant gene editing through de novo induction of meristems. Nat. Biotechnol. 38, 84–89 (2020). This work expressed developmental regulators and targeted genome-modification reagents, leading to de novo induction of meristematic tissues and edited plants obtained without the need for tissue culture.
Oh, Y., Kim, H. & Kim, S. G. Virus-induced plant genome editing. Curr. Opin. Plant. Biol. 60, 101992 (2021).
Ellison, E. E. et al. Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs. Nat. Plants 6, 620–624 (2020). This study used an RNA virus to express mobile single guide RNA, enabling efficient multiplex genome editing in plants without the need for tissue culture.
Čermák, T. et al. A multipurpose toolkit to enable advanced genome engineering in plants. Plant. Cell 29, 1196–1217 (2017).
Li, T. et al. Highly efficient heritable genome editing in wheat using an RNA virus and bypassing tissue culture. Mol. Plant. 14, 1787–1798 (2021).
Ma, X., Zhang, X., Liu, H. & Li, Z. Highly efficient DNA-free plant genome editing using virally delivered CRISPR–Cas9. Nat. Plants 6, 773–779 (2020).
Liu, Q., Zhao, C., Sun, K., Deng, Y. & Li, Z. Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes. Mol. Plant. 16, 616–631 (2023).
Park, S. Y., Shimizu, K., Brown, J., Aoki, K. & Westwood, J. H. Mobile host mRNAs are translated to protein in the associated parasitic plant Cuscuta campestris. Plants 11, 93 (2021).
Zhang, W. et al. tRNA-related sequences trigger systemic mRNA transport in plants. Plant Cell 28, 1237–1249 (2016).
Jackson, S. D. & Hong, Y. Systemic movement of FT mRNA and a possible role in floral induction. Front. Plant. Sci. 3, 127 (2012).
Yang, L., Machin, F., Wang, S., Saplaoura, E. & Kragler, F. Heritable transgene-free genome editing in plants by grafting of wild-type shoots to transgenic donor rootstocks. Nat. Biotechnol. 41, 958–967 (2023). This work reported the first transgene-free and tissue-culture-free plant delivery by grafting, achieving heritable targeted mutagenesis.
Woo, J. W. et al. DNA-free genome editing in plants with preassembled CRISPR–Cas9 ribonucleoproteins. Nat. Biotechnol. 33, 1162–1164 (2015).
Andersson, M. et al. Genome editing in potato via CRISPR–Cas9 ribonucleoprotein delivery. Physiol. Plant. 164, 378–384 (2018).
Liu, Y. et al. Establishment of a DNA-free genome editing and protoplast regeneration method in cultivated tomato (Solanum lycopersicum). Plant. Cell Rep. 41, 1843–1852 (2022).
Cao, X. et al. Cut-dip-budding delivery system enables genetic modifications in plants without tissue culture. Innovation 4, 100345 (2023).
Raman, V. et al. Agrobacterium expressing a type III secretion system delivers Pseudomonas effectors into plant cells to enhance transformation. Nat. Commun. 13, 2581 (2022).
Soliman, A., Laurie, J., Bilichak, A. & Ziemienowicz, A. Applications of CPPs in genome editing of plants. Methods Mol. Biol. 2383, 595–616 (2022).
Kwak, S. Y. et al. Chloroplast-selective gene delivery and expression in planta using chitosan-complexed single-walled carbon nanotube carriers. Nat. Nanotechnol. 14, 447–455 (2019).
Wang, Z. P. et al. Efficient and genotype independent maize transformation using pollen transfected by DNA-coated magnetic nanoparticles. J. Integr. Plant. Biol. 64, 1145–1156 (2022).
Ran, Y., Liang, Z. & Gao, C. Current and future editing reagent delivery systems for plant genome editing. Sci. China Life Sci. 60, 490–505 (2017).
Li, C. et al. Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors. Nat. Biotechnol. 38, 875–882 (2020). This work generated dual cytosine and adenine base editors as mutagenesis editors that achieved near-saturated mutagenesis and facilitated directed evolution of plant endogenous genes.
Kuang, Y. et al. Base-editing-mediated artificial evolution of OsALS1 in planta to develop novel herbicide-tolerant rice germplasms. Mol. Plant. 13, 565–572 (2020).
Zhang, A. et al. Directed evolution rice genes with randomly multiplexed sgRNAs assembly of base editors. Plant Biotechnol. J. 21, 2597–2610 (2023).
Zhang, C. et al. Artificial evolution of OsEPSPS through an improved dual cytosine and adenine base editor generated a novel allele conferring rice glyphosate tolerance. J. Integr. Plant. Biol. 65, 2194–2203 (2023).
Xu, R., Liu, X., Li, J., Qin, R. & Wei, P. Identification of herbicide resistance OsACC1 mutations via in planta prime-editing-library screening in rice. Nat. Plants 7, 888–892 (2021).
Romero, I. G., Ruvinsky, I. & Gilad, Y. Comparative studies of gene expression and the evolution of gene regulation. Nat. Rev. Genet. 13, 505–516 (2012).
Liu, L. et al. Enhancing grain-yield-related traits by CRISPR–Cas9 promoter editing of maize CLE genes. Nat. Plants 7, 287–294 (2021).
Cui, Y., Cao, Q., Li, Y., He, M. & Liu, X. Advances in cis-element- and natural variation-mediated transcriptional regulation and applications in gene editing of major crops. J. Exp. Bot. 74, 5441–5457 (2023).
Zhou, J. et al. An efficient CRISPR–Cas12a promoter editing system for crop improvement. Nat. Plants 9, 588–604 (2023).
Rodriguez-Leal, D., Lemmon, Z. H., Man, J., Bartlett, M. E. & Lippman, Z. B. Engineering quantitative trait variation for crop improvement by genome editing. Cell 171, 470–480.e478 (2017). This work demonstrates that targeting cis-regulatory elements in promoter regions can create a continuum of variation in genes for improving crop traits.
Aguirre, L., Hendelman, A., Hutton, S. F., McCandlish, D. M. & Lippman, Z. B. Idiosyncratic and dose-dependent epistasis drives variation in tomato fruit size. Science 382, 315–320 (2023).
Song, X. et al. Targeting a gene regulatory element enhances rice grain yield by decoupling panicle number and size. Nat. Biotechnol. 40, 1403–1411 (2022).
Wang, J., Liu, J. & Guo, Z. Natural uORF variation in plants. Trends Plant Sci. https://doi.org/10.1016/j.tplants.2023.07.005 (2023).
Zhang, H. et al. Genome editing of upstream open reading frames enables translational control in plants. Nat. Biotechnol. 36, 894–898 (2018). This study demonstrates a strategy that manipulates crop quantitative traits by fine-tuning protein expression through the modification of upstream open reading frames.
Xing, S. et al. Fine-tuning sugar content in strawberry. Genome Biol. 21, 230 (2020).
Xue, C. et al. Tuning plant phenotypes by precise, graded downregulation of gene expression. Nat. Biotechnol. 41, 1758–1764 (2023).
Wang, H. et al. Genome editing of 3′ UTR-embedded inhibitory region enables generation of gene knock-up alleles in plants. Plant. Commun. https://doi.org/10.1016/j.xplc.2023.100745 (2023).
Meng, F. et al. Genomic editing of intronic enhancers unveils their role in fine-tuning tissue-specific gene expression in Arabidopsis thaliana. Plant. Cell 33, 1997–2014 (2021).
Guo, N. et al. Genome sequencing sheds light on the contribution of structural variants to Brassica oleracea diversification. BMC Biol. 19, 93 (2021).
Alonge, M. et al. Major impacts of widespread structural variation on gene expression and crop improvement in tomato. Cell 182, 145–161.e123 (2020).
Lu, Y. et al. A donor-DNA-free CRISPR/Cas-based approach to gene knock-up in rice. Nat. Plants 7, 1445–1452 (2021).
Li, S. et al. Genome-edited powdery mildew resistance in wheat without growth penalties. Nature 602, 455–460 (2022). This study utilized multiplexed genome editing to improve complex polyploid crops, while achieving a dual benefit of high yield and disease resistance.
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).
Zhou, Y. et al. Graph pangenome captures missing heritability and empowers tomato breeding. Nature 606, 527–534 (2022).
Zhang, J., Yu, H. & Li, J. De novo domestication: retrace the history of agriculture to design future crops. Curr. Opin. Biotechnol. 81, 102946 (2023).
Li, T. et al. Domestication of wild tomato is accelerated by genome editing. Nat. Biotechnol. 36, 1160–1163 (2018).
Yu, H. et al. A route to de novo domestication of wild allotetraploid rice. Cell 184, 1156–1170.e1114 (2021).
Lemmon, Z. H. et al. Rapid improvement of domestication traits in an orphan crop by genome editing. Nat. Plants 4, 766–770 (2018).
Zsögön, A. et al. De novo domestication of wild tomato using genome editing. Nat. Biotechnol. 36, 1211–1216 (2018). This work is the first to demonstrate that genome editing can rapidly generate novel crops by de novo domestication of wild plant species.
Smýkal, P., Nelson, M., Berger, J. & Von Wettberg, E. The impact of genetic changes during crop domestication. Agronomy 8, 26 (2018).
Huang, X., Huang, S., Han, B. & Li, J. The integrated genomics of crop domestication and breeding. Cell 185, 2828–2839 (2022).
DeHaan, L. et al. Roadmap for accelerated domestication of an emerging perennial grain crop. Trends Plant. Sci. 25, 525–537 (2020).
Okada, A. et al. CRISPR/Cas9-mediated knockout of Ms1 enables the rapid generation of male-sterile hexaploid wheat lines for use in hybrid seed production. Plant Biotechnol. J. 17, 1905–1913 (2019).
Singh, M., Kumar, M., Albertsen, M. C., Young, J. K. & Cigan, A. M. Concurrent modifications in the three homeologs of Ms45 gene with CRISPR–Cas9 lead to rapid generation of male sterile bread wheat (Triticum aestivum L.). Plant. Mol. Biol. 97, 371–383 (2018).
Chen, G. et al. Gene editing to facilitate hybrid crop production. Biotechnol. Adv. 46, 107676 (2021).
Vernet, A. et al. High-frequency synthetic apomixis in hybrid rice. Nat. Commun. 13, 7963 (2022).
Marimuthu, M. P. et al. Synthetic clonal reproduction through seeds. Science 331, 876 (2011).
Wang, C. et al. Clonal seeds from hybrid rice by simultaneous genome engineering of meiosis and fertilization genes. Nat. Biotechnol. 37, 283–286 (2019).
Wei, X. et al. Synthetic apomixis with normal hybrid rice seed production. Mol. Plant. 16, 489–492 (2023).
Khanday, I., Skinner, D., Yang, B., Mercier, R. & Sundaresan, V. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds. Nature 565, 91–95 (2019). This work achieved apomixis in rice with the assistance of targeted genome modification, which can be utilized to fix hybrid vigour.
Song, M. et al. Simultaneous production of high-frequency synthetic apomixis with high fertility and improved agronomic traits in hybrid rice. Mol. Plant. 17, 4–7 (2024).
Kelliher, T. et al. MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction. Nature 542, 105–109 (2017).
Liu, C. et al. A 4-bp insertion at ZmPLA1 encoding a putative phospholipase A generates haploid induction in maize. Mol. Plant. 10, 520–522 (2017).
Lv, J. et al. Generation of paternal haploids in wheat by genome editing of the centromeric histone CENH3. Nat. Biotechnol. 38, 1397–1401 (2020).
Li, Y. et al. Loss-of-function alleles of ZmPLD3 cause haploid induction in maize. Nat. Plants 7, 1579–1588 (2021).
Zhong, Y. et al. Mutation of ZmDMP enhances haploid induction in maize. Nat. Plants 5, 575–580 (2019).
Jiang, C. et al. A reactive oxygen species burst causes haploid induction in maize. Mol. Plant. 15, 943–955 (2022).
Kelliher, T. et al. One-step genome editing of elite crop germplasm during haploid induction. Nat. Biotechnol. 37, 287–292 (2019). This work integrated haploid induction technology with genome editing, and generated the HI-Edit system, thus achieving rapid improvement of a wide range of crop varieties.
Wang, B. et al. Development of a haploid-inducer-mediated genome-editing system for accelerating maize breeding. Mol. Plant. 12, 597–602 (2019).
Ye, M. et al. Generation of self-compatible diploid potato by knockout of S-RNase. Nat. Plants 4, 651–654 (2018).
Ma, C. et al. CRISPR/Cas9-mediated multiple gene editing in Brassica oleracea var. capitata using the endogenous tRNA-processing system. Hortic. Res. 6, 20 (2019).
Srivastava, V. & Thomson, J. Gene stacking by recombinases. Plant Biotechnol. J. 14, 471–482 (2016).
Zhu, Q. et al. Development of ‘purple endosperm rice’ by engineering anthocyanin biosynthesis in the endosperm with a high-efficiency transgene stacking system. Mol. Plant. 10, 918–929 (2017).
Shehryar, K. et al. Transgene stacking as effective tool for enhanced disease resistance in plants. Mol. Biotechnol. 62, 1–7 (2020).
Glasscock, C. J. et al. Computational design of sequence-specific DNA-binding proteins. Preprint at bioRxiv https://doi.org/10.1101/2023.09.20.558720 (2023).
Watson, J. L. et al. De novo design of protein structure and function with RFdiffusion. Nature 620, 1089–1100 (2023).
Xu, C. et al. Computational design of transmembrane pores. Nature 585, 129–134 (2020).
Renard, D. & Tilman, D. National food production stabilized by crop diversity. Nature 571, 257–260 (2019).
Shalev, O. et al. Commensal Pseudomonas strains facilitate protective response against pathogens in the host plant. Nat. Ecol. Evol. 6, 383–396 (2022).
Xu, S. et al. Fusarium fruiting body microbiome member Pantoea agglomerans inhibits fungal pathogenesis by targeting lipid rafts. Nat. Microbiol. 7, 831–843 (2022).
Jiang, D. et al. Highly efficient genome editing in Xanthomonas oryzae pv. oryzae through repurposing the endogenous type I‐C CRISPR–Cas system. Mol. Plant. Pathol. 23, 583–594 (2021).
Zhang, X.-E. et al. Enabling technology and core theory of synthetic biology. Sci. China Life Sci. 66, 1742–1785 (2023).
Vora, Z., Pandya, J., Sangh, C. & Vaikuntapu, P. R. The evolving landscape of global regulations on genome-edited crops. J. Plant. Biochem. Biotechnol. 32, 831–845 (2023).
Ahmad, A., Jamil, A. & Munawar, N. GMOs or non-GMOs? The CRISPR conundrum. Front. Plant. Sci. 14, 1232938 (2023).
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).
Chen, Y. et al. CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis. Sci. China Life Sci. 60, 520–523 (2017).
Tian, S. et al. Engineering herbicide-resistant watermelon variety through CRISPR/Cas9-mediated base-editing. Plant. Cell Rep. 37, 1353–1356 (2018).
Qin, L. et al. High‐efficient and precise base editing of C•G to T•A in the allotetraploid cotton (Gossypium hirsutum) genome using a modified CRISPR/Cas9 system. Plant Biotechnol. J. 18, 45–56 (2019).
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).
Shimatani, Z. et al. Targeted base editing in rice and tomato using a CRISPR–Cas9 cytidine deaminase fusion. Nat. Biotechnol. 35, 441–443 (2017).
Ren, B. et al. Improved base editor for efficiently inducing genetic variations in rice with CRISPR/Cas9-guided hyperactive hAID mutant. Mol. Plant. 11, 623–626 (2018).
Wang, M. et al. Optimizing base editors for improved efficiency and expanded editing scope in rice. Plant Biotechnol. J. 17, 1697–1699 (2019).
Hua, K., Tao, X. & Zhu, J. K. Expanding the base editing scope in rice by using Cas9 variants. Plant Biotechnol. J. 17, 499–504 (2019).
Ren, B. et al. Cas9–NG greatly expands the targeting scope of the genome-editing toolkit by recognizing NG and other atypical PAMs in rice. Mol. Plant. 12, 1015–1026 (2019).
Veillet, F. et al. The Solanum tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid potato. Plant. Cell Rep. 38, 1065–1080 (2019).
Veillet, F. et al. Transgene-free genome editing in tomato and potato plants using Agrobacterium-mediated delivery of a CRISPR/Cas9 cytidine base editor. Int. J. Mol. Sci. 20, 402 (2019).
Wu, J. et al. Engineering herbicide‐resistant oilseed rape by CRISPR/Cas9‐mediated cytosine base‐editing. Plant Biotechnol. J. 18, 1857–1859 (2020).
Wang, M. et al. Targeted base editing in rice with CRISPR/ScCas9 system. Plant Biotechnol. J. 18, 1645–164 (2020).
Zhang, C. et al. Expanding the base editing scope to GA and relaxed NG PAM sites by improved xCas9 system. Plant Biotechnol. J. 18, 884–886 (2019).
Zeng, D. et al. PhieCBEs: plant high-efficiency cytidine base editors with expanded target range. Mol. Plant. 13, 1666–1669 (2020).
Yan, F. et al. Highly efficient A·T to G·C base editing by Cas9n-guided tRNA adenosine deaminase in rice. Mol. Plant. 11, 631–634 (2018).
Hu, L. et al. Precise A∙T to G∙C base editing in the allotetraploid rapeseed (Brassica napus L.) genome. J. Cell Physiol. 237, 4544–4550 (2022).
Hua, K. et al. Simplified adenine base editors improve adenine base editing efficiency in rice. Plant Biotechnol. J. 18, 770–778 (2019).
Yan, D. et al. High-efficiency and multiplex adenine base editing in plants using new TadA variants. Mol. Plant. 14, 722–731 (2021).
Tan, J. et al. PhieABEs: a PAM‐less/free high‐efficiency adenine base editor toolbox with wide target scope in plants. Plant Biotechnol. J. 20, 934–943 (2022).
Xu, R. et al. Development of an efficient plant dual cytosine and adenine editor. J. Integr. Plant. Biol. 63, 1600–1605 (2021).
Tian, Y. et al. Efficient C‐to‐G editing in rice using an optimized base editor. Plant. Biotechnol. 20, 1238–1240 (2022).
Cheng, Y. et al. CRISPR–Cas12a base editors confer efficient multiplexed genome editing in rice. Plant. Commun. 4, 100601 (2023).
Wang, D. et al. Developing a highly efficient CGBE base editor in watermelon. Hortic. Res. 10, uhad155 (2023).
Li, Y. et al. Engineering a plant A-to-K base editor with improved performance by fusion with a transactivation module. Plant. Commun. 4, 100667 (2023).
Li, X. et al. Efficient and heritable A-to-K base editing in rice and tomato. Hortic. Res. 11, uhad250 (2024).
Zhong, D. et al. Targeted A‐to‐T and A‐to‐C base replacement in maize using an optimized adenine base editor. Plant Biotechnol. J. 22, 541–543 (2023).
Lin, Q. et al. Prime genome editing in rice and wheat. Nat. Biotechnol. 38, 582–585 (2020).
Lin, Q. et al. High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat. Biotechnol. 39, 923–927 (2021).
Butt, H. et al. Engineering herbicide resistance via prime editing in rice. Plant Biotechnol. J. 18, 2370–2372 (2020).
Hua, K., Jiang, Y., Tao, X. & Zhu, J. K. Precision genome engineering in rice using prime editing system. Plant Biotechnol. J. 18, 2167–2169 (2020).
Xu, R. et al. Development of plant prime-editing systems for precise genome editing. Plant. Commun. 1, 100043 (2020).
Jiang, Y.-Y. et al. Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize. Genome Biol. 21, 257 (2020).
Lu, Y. et al. Precise genome modification in tomato using an improved prime editing system. Plant Biotechnol. J. 19, 415–417 (2020).
Li, J. et al. Development of a highly efficient prime editor 2 system in plants. Genome Biol. 23, 161 (2022).
Li, H. et al. Multiplex precision gene editing by a surrogate prime editor in rice. Mol. Plant. 15, 1077–1080 (2022).
Zou, J. et al. Improving the efficiency of prime editing with epegRNAs and high-temperature treatment in rice. Sci. China Life Sci. 65, 2328–2331 (2022).
Liang, Z., Wu, Y., Guo, Y. & Wei, S. Addition of the T5 exonuclease increases the prime editing efficiency in plants. J. Genet. Genom. 50, 582–588 (2023).
Gupta, A., Liu, B., Raza, S., Chen, Q.-J. & Yang, B. Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice. Plant Commun. 5, 100471 (2023).
Gupta, A., Liu, B., Chen, Q. J. & Yang, B. High‐efficiency prime editing enables new strategies for broad‐spectrum resistance to bacterial blight of rice. Plant Biotechnol. J. 21, 1454–1464 (2023).
Liu, X. et al. Generating herbicide resistant and dwarf rice germplasms through precise sequence insertion or replacement. Plant Biotechnol. J. 22, 293–295 (2023).
Wang, J. et al. Plant organellar genomes: much done, much more to do. Trends Plant. Sci. https://doi.org/10.1016/j.tplants.2023.12.014 (2024).
Kang, B.-C. et al. Chloroplast and mitochondrial DNA editing in plants. Nat. Plants 7, 899–905 (2021).
Kazama, T. et al. Curing cytoplasmic male sterility via TALEN-mediated mitochondrial genome editing. Nat. Plants 5, 722–730 (2019).
Kuwabara, K., Arimura, S.-i, Shirasawa, K. & Ariizumi, T. orf137 triggers cytoplasmic male sterility in tomato. Plant. Physiol. 189, 465–468 (2022).
Mok, B. Y. et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature 583, 631–63 (2020).
Nakazato, I. et al. Targeted base editing in the plastid genome of Arabidopsis thaliana. Nat. Plants 7, 906–913 (2021).
Hu, J. et al. Strand-preferred base editing of organellar and nuclear genomes using CyDENT. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01910-9 (2023).
Mok, Y. G., Hong, S., Bae, S. J., Cho, S. I. & Kim, J. S. Targeted A-to-G base editing of chloroplast DNA in plants. Nat. Plants 8, 1378–1384 (2022).
Yi, Z. et al. Strand-selective base editing of human mitochondrial DNA using mitoBEs. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01791-y (2023).
Acknowledgements
This work was supported by the National Natural Science Foundation of China (32388201), the National Key Research and Development Program (2022YFF1002802), the Ministry of Agriculture and Rural Affairs of China, the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24020102), and the New Cornerstone Science Foundation. The authors thank K. T. Zhao, C. Xue, R. Liang, G. Liu, J. Hu, H. Li, Y. Li, F. Qiu, S. Li, Y. Lei and X. Jiang for their insightful comments on the manuscript.
Author information
Authors and Affiliations
Contributions
B.L., C.S. and C.G. researched the literature. All authors substantially contributed to discussions of the content and wrote the article. J.L. and C.G. reviewed and/or edited the manuscript before submission.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Reviews Genetics thanks José R. Botella, who co-reviewed with Zheng (Tommy) Gong; Yuriko Osakabe; and Yiping Qi for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Glossary
- Agrobacterium rhizogenes
-
A bacterium used for plant delivery. It can induce the formation of hairy roots in the infection site. It contains a root-inducing plasmid that carries a T-DNA segment capable of integrating into the plant genome. Typically, this T-DNA harbours the desired sequences intended for transfer into the plant genome.
- Agrobacterium tumefaciens
-
A bacterium used for plant delivery. It contains a modified tumour-inducing plasmid that carries a T-DNA segment capable of integrating into the plant genome. Typically, this T-DNA harbours the desired sequences intended for transfer into the plant genome, as well as marker genes for selecting positive events.
- CRISPR interference
-
(CRISPRi). CRISPR interference utilizes dCas9 either alone or with a transcription repressor to inhibit gene expression by targeting specific DNA sequences without altering the genetic code, offering precise control for studying gene functions and regulatory processes within cells.
- Guide RNA
-
(gRNA). An RNA molecule used to direct Cas9 or similar enzymes to a specific DNA or RNA sequence for precise modification.
- Hybrid vigour
-
A phenomenon in which the offspring of two different inbred lines or varieties exhibit improved traits compared to their parents, such as increased yield, growth or biotic or abiotic resistance. Also known as heterosis.
- Non-targeted strand
-
The DNA strand that is not complementary to the guide RNA sequence. DNA nicking by PE2 and base deamination by base editors occur on the non-targeted DNA strand.
- Particle bombardment
-
A genetic transformation technique, also known as gene gun or biolistic delivery, that involves loading exogenous DNA onto microscopic metal particles that are accelerated and propelled into plant cells or other target cells by compressed gas or physical force.
- Protospacer adjacent motif
-
(PAM). A short DNA sequence immediately adjacent to the target site that is essential for the recognition and binding of Cas protein to the target DNA.
- R-loop
-
A specific structure consisting of one DNA strand, its complementary DNA strand and an RNA strand located between them.
- Targeted strand
-
The DNA strand that is complementary to the guide RNA sequence. DNA nicking by base editors occurs on the targeted DNA strand.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Li, B., Sun, C., Li, J. et al. Targeted genome-modification tools and their advanced applications in crop breeding. Nat Rev Genet (2024). https://doi.org/10.1038/s41576-024-00720-2
Accepted:
Published:
DOI: https://doi.org/10.1038/s41576-024-00720-2
