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non-transgenic Plant Genome Editing Using Purified Sequence-Specific Nucleases

Molecular Plant 2015年第9期8卷 1425-1427页

作者： Song Luo Jin Li Thomas J. Stoddard Nicholas J. Baltes Zachary L. Demorest Benjamin M. Clasen Andrew Coffman Adam Retterath Luc Mathis Daniel F. Voytas Feng Zhang Cellectis Plant Sciences 600 County Road D West Suite 8 New Brighton MN 55112 USA

Dear Editor Sequence-specific nucleases, including zinc-finger nucleases, meganucleases, TAL effector nucleases （TALENs）, and CRISPR/ Cas systems, have been used to introduce targeted mutations in a wide range of plant species （Voytas, 2013; Baltes and Voytas, 2015）. However, delivery of these nucleases using traditional transformation methods （e.g., particle bombardment, Agrobacterium or protoplast transformation） may result in undesired genetic alterations due to random insertion of nucleaseencoding DNA into the host genome. Random integration of DNA is a particular concern for trait improvement and gene function studies, because it may lead to unintended gene inactivation or it may alter expression of host genes. Furthermore, for crop varieties created using biotechnology, the presence of foreign DNA is a trigger for regulation by many governmental agencies （Voytas and Gao, 2014）. Therefore, methods to modify plant genomes that do not require DNA delivery would have value in both commercial and academic settings. Here we demonstrate nontransgenic plant genome engineering by introducing sequencespecific nucleases as purified protein. This approach enabled targeted mutagenesis of endogenous sequences within plant cells, while avoiding integration of foreign DNA into the genome.

关键词： non-transgenic Plant Genome

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Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes

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Molecular Plant 2023年第3期16卷 616-631页

作者： Qian Liu Chenglu Zhao Kai Sun Yinlu Deng Zhenghe Li State Key Laboratory of Rice Biology Institute of BiotechnologyZhejang UniversityHangzhouChina Ministry of Agricutture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests Zhejiang UniversityHangzhouChina Key Laboratory of Biology of Crop Pathogens and Insects of Zhejang Province Zhejang UniversityHangzhouChina

CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop ***,the lack of robust delivery methods has limited the widespread adoption of these revolutionary technologies in plant ***,we report an efficient,non-transgenic CRISPR/Cas delivery platform based on the engineered tomato spotted wilt virus(TSWV),an RNA virus with a host range of over 1000 plant *** eliminated viral elements essential for insect transmission to liberate genome space for accommodating large genetic cargoes without sacrificing the ability to infect plant *** resulting non-insect-transmissible viral vectors enabled effective and stable in planta delivery of Cas12a and Cas9 nucleases as well as adenine and cytosine base *** systemically infected plant tissues,the deconstructed TSWV-derived vectors induced efficient somatic gene mutations and base conversions in multiple crop species with little genotype *** with heritable,bi-allelic mutations could be readily regenerated by culturing the virus-infected tissues in vitro without antibiotic ***,we showed that antiviral treatment with ribavirin during tissue culture cleared the viral vectors in 100%of regenerated plants and further augmented the recovery of heritable *** many plants are recalcitrant to stable transformation,the viral delivery system developed in this work provides a promising tool to overcome gene delivery bottlenecks for genome editing in various crop species and elite varieties.

关键词： genome editing CRISPR-Cas base editor RNA virus vector tomato spotted wilt virus bunyavirus delivery non-transgenic

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