Plant Genome Editing Technologies
Plant genome editing relies on programmable sequence-specific nucleases (SSNs), including engineered homing endonucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas system. These SSNs induce DNA double-strand breaks at target sites, enabling precise genome modifications through DNA repair pathways. The CRISPR-Cas system, known for its cost-effectiveness and simplicity, has become the predominant tool in plant genome editing.
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Fig. 1 General procedure for plant genome editing (Gao C., 2021).
The general procedure for plant genome editing involves six steps: (1) selecting the appropriate nuclease, (2) constructing genome editing vectors, (3) validating vector activity using protoplasts, (4) delivering editing reagents into plant cells, (5) regenerating edited cells into plantlets, and (6) screening and genotyping edited plants. While the same editing tools are used across organisms, plant-specific challenges arise in delivery and regeneration.
Next-Generation Plant Breeding Techniques
Traditional plant breeding faces constraints in feeding the growing global population. Advances like marker-assisted selection and genomics-assisted breeding push boundaries. Genome editing accelerates plant breeding, enabling unprecedented speed and efficiency. This technology propels plant breeding to the next generation, surpassing current limits.
Haploid Induction: A Leap in Breeding Efficiency
Traditional plant breeding methods often involve six to seven generations of self-pollination to produce highly homozygous and stable cultivars. Haploid induction, facilitated by CRISPR-Cas9 technology, offers a game-changing alternative. The knockout of genes such as MTL/PLA1/NLD and DMP has been demonstrated to induce defective male gametophytes, leading to maternal haploid induction in maize, rice, and wheat. Additionally, targeted editing of CENH3 in A. thaliana and TaCENH3α in wheat has resulted in the development of haploid inducer lines, further streamlining the breeding process.
Artificial Apomixis: Toward Clonal Seeds
Apomixis, the natural process of producing genetically identical seeds, has been a focus of engineering efforts. CRISPR-Cas9 has been instrumental in inducing apomeiosis in rice by targeting meiotic genes such as REC8, PAIR1, and OSD1. Moreover, synthetic apomixis has been achieved by manipulating the expression of BBM1 in unfertilized egg cells, demonstrating the potential for generating clonal seeds in various crops.
Large-Scale Screening for Trait Discovery
Understanding the genetic regulation of beneficial traits is paramount for effective genome editing in plant breeding. CRISPR-Cas9 screens serve as powerful tools for genome-wide characterization, allowing the identification of genes associated with desirable phenotypes. Large-scale screening, facilitated by the expression of Cas9 with a library of sgRNAs targeting multiple genes, has proven effective in rice, tomato, soybean, and maize. This approach, coupled with high-throughput phenotyping, is instrumental in discovering traits that can contribute to crop improvement.
Directed Evolution for Trait Engineering
Another avenue explored in large-scale screening involves the introduction of saturated mutations in a gene or its functional domain, followed by directed evolution. CRISPR-Cas9 has been successfully employed in directed protein evolution, utilizing a library of sgRNAs to induce mutations and select plants with desired properties. This approach, when combined with base editing systems like CBE and ABE, holds promise for achieving near-saturation mutagenesis of target domains.
Challenges and Future Perspectives
Despite the progress in genome engineering, challenges persist. Improving the efficiency of precise genome editing, enhancing specificity to minimize off-target effects, and optimizing plant cell delivery and regeneration systems are ongoing endeavors. HDR-mediated genome editing holds the potential for precise changes, but its efficiency in somatic plant cells remains a challenge1. The development of new base editing systems and prime editing, along with advancements in plant cell delivery methods, is crucial to overcoming these limitations.
Plant Synthetic Biology: Designing the Future
Synthetic biology, empowered by CRISPR-Cas systems, offers a strategic approach to plant design. Multiplex gene editing and regulation enable the redirection of metabolic networks, leading to the production of crops enriched in desired compounds. Beyond trait improvement, genome editing facilitates the optimization of photosynthesis systems, exemplified by efforts to enhance Rubisco efficiency and redesign Rubisco itself.
Plant Microbiome Engineering: Unraveling the Second Genome
Recognizing the importance of the plant microbiome, genome engineering is extending its reach to precisely edit microbial communities associated with plants. This "second genome" influences plant growth, nutrient absorption, and pathogen resistance. CRISPR-Cas technology allows for the targeted editing of specific organisms within microbial communities, offering new insights into their roles and functions. The potential for in situ genome editing of plant microbiomes opens new avenues for improving crop production.
In conclusion, genome engineering stands at the forefront of a new era in agriculture, offering unparalleled opportunities for crop improvement and sustainable food production. The combination of haploid induction, artificial apomixis, large-scale screening, and synthetic biology holds the promise of a second Green Revolution. However, challenges in precise editing efficiency, specificity, and delivery systems must be addressed collaboratively by researchers, policymakers, and industry stakeholders. Genome editing, integrated with other technologies, is poised to revolutionize plant breeding and contribute significantly to meeting the food demands of a growing global population under changing climate conditions.
Reference
- Gao C. Genome engineering for crop improvement and future agriculture. Cell. 2021, 184(6): 1621-1635.
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