Highly efficient DNA-free plant genome editing using virally delivered CRISPR-Cas9
Highly efficient DNA-free plant genome editing using virally delivered CRISPR-Cas9
复制标题
使用病毒传递的 CRISPR-Cas9 进行高效的无 DNA 植物基因组编辑
DOI：
10.1038/s41477-020-0704-5
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发表时间：
2020-06-29
期刊：
影响因子：
18
通讯作者：
Li, Zhenghe
中科院分区：
文献类型：
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作者：
Ma, Xiaonan;Zhang, Xiaoyan;Li, Zhenghe
使用CRISPR-Cas核酸酶的基因组编辑技术已经彻底改变了植物科学，并在作物改良方面具有巨大的前景。传统的转基因介导的CRISPR-Cas试剂递送方法可能与意想不到的基因组变化或损伤（1,2）、涉及外源DNA分离的育种周期延长以及转基因方面的监管限制有关(3)。因此，通过将预组装的CRISPR-Cas9核糖核蛋白转染原生质体(4)或体外受精受精卵(5)，可以实现无dna递送。然而，从这些无壁细胞中再生的技术困难使得这些方法在大多数作物物种中普遍适用不切实际。另外，CRISPR-Cas核糖核蛋白或RNA转录物已经被生物轰击到未成熟的胚胎细胞或愈伤组织中，以产生高度特异性的基因组编辑，尽管频率很低（6-9）。在这里，我们报道了一种基于植物负链RNA病毒的载体的工程设计，用于在植物中无dna递送整个CRISPR-Cas9盒，以实现异源四倍体模型烟草宿主的单、多重诱变和高频染色体缺失。在未经选择的病毒感染组织再生的植株中，超过90%含有靶向突变，其中高达57%携带四等位基因的可遗传突变。即使经过机械传播，病毒载体仍然保持稳定，并且可以在再生或结实子后很容易地从突变植株中消除。尽管有很高的目标活动，偏离目标的影响，如果有的话，是最小的。我们的研究为通过病毒感染对植物进行CRISPR-Cas9基因编辑提供了一种方便、高效、经济的方法。开发了一种基于RNA病毒感染的植物无dna基因编辑方法，允许将整个CRISPR-Cas9磁带体递送到烟草宿主中，实现高效的单、多重诱变和染色体缺失。
Genome-editing technologies using CRISPR-Cas nucleases have revolutionized plant science and hold enormous promise in crop improvement. Conventional transgene-mediated CRISPR-Cas reagent delivery methods may be associated with unanticipated genome changes or damage(1,2), with prolonged breeding cycles involving foreign DNA segregation and with regulatory restrictions regarding transgenesis(3). Therefore, DNA-free delivery has been developed by transfecting preassembled CRISPR-Cas9 ribonucleoproteins into protoplasts(4)or in vitro fertilized zygotes(5). However, technical difficulties in regeneration from these wall-less cells make impractical a general adaption of these approaches to most crop species. Alternatively, CRISPR-Cas ribonucleoproteins or RNA transcripts have been biolistically bombarded into immature embryo cells or calli to yield highly specific genome editing, albeit at low frequency(6-9). Here we report the engineering of a plant negative-strand RNA virus-based vector for DNA-free in planta delivery of the entire CRISPR-Cas9 cassette to achieve single, multiplex mutagenesis and chromosome deletions at high frequency in a model allotetraploid tobacco host. Over 90% of plants regenerated from virus-infected tissues without selection contained targeted mutations, among which up to 57% carried tetra-allelic, inheritable mutations. The viral vector remained stable even after mechanical transmission, and can readily be eliminated from mutated plants during regeneration or after seed setting. Despite high on-target activities, off-target effects, if any, are minimal. Our study provides a convenient, highly efficient and cost-effective approach for CRISPR-Cas9 gene editing in plants through virus infection.A DNA-free in planta approach for gene editing based on RNA virus infection is developed, allowing delivery of the entire CRISPR-Cas9 cassettes into tobacco host to achieve highly efficient single, multiplex mutagenesis and chromosome deletions.