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  • Review Article
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Targeted genome-modification tools and their advanced applications in crop breeding

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.

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Fig. 1: Precise DNA editing at the base pair level.
Fig. 2: Precise editing of large DNA segments.
Fig. 3: Delivery technologies used in plant targeted genome modification.
Fig. 4: Advanced applications of TGM in crop breeding.
Fig. 5: Prospects for TGM technologies in crop breeding.

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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.

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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.

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Correspondence to Caixia Gao.

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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.

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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

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