Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes
作者机构：State Key Laboratory of Rice BiologyInstitute of BiotechnologyZhejang UniversityHangzhouChina Ministry of Agricutture Key Laboratory of Molecular Biology of Crop Pathogens and Insect PestsZhejiang UniversityHangzhouChina Key Laboratory of Biology of Crop Pathogens and Insects of Zhejang ProvinceZhejang UniversityHangzhouChina
出 版 物：《Molecular Plant》 (分子植物（英文版）)
年 卷 期：2023年第16卷第3期
页 面：616-631页
核心收录：
学科分类：0710[理学-生物学] 071001[理学-植物学] 07[理学]
基 金：supported by grants from the National Key R&D Program of China(2022YFC2601000) the Natural Science Foundation of Zhejiang Province,China(no.LZ20C140004) the National Natural Science Foundation of China(no.31870142).
主 题：genome editing CRISPR-Cas base editor RNA virus vector tomato spotted wilt virus bunyavirus delivery non-transgenic
摘 要：CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop breeding.However,the lack of robust delivery methods has limited the widespread adoption of these revolutionary technologies in plant science.Here,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 species.We eliminated viral elements essential for insect transmission to liberate genome space for accommodating large genetic cargoes without sacrificing the ability to infect plant hosts.The 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 editors.In systemically infected plant tissues,the deconstructed TSWV-derived vectors induced efficient somatic gene mutations and base conversions in multiple crop species with little genotype dependency.Plants with heritable,bi-allelic mutations could be readily regenerated by culturing the virus-infected tissues in vitro without antibiotic selection.Moreover,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 mutations.Because 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.
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