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Highly efficient DNA-free plant genome editing using virally delivered CRISPR-Cas9.
Nature Plants ( IF 18.0 ) Pub Date : 2020-06-29 , DOI: 10.1038/s41477-020-0704-5
Xiaonan Ma 1 , Xiaoyan Zhang 1 , Huimin Liu 1 , Zhenghe Li 1, 2, 3
Affiliation  

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 damage1,2, with prolonged breeding cycles involving foreign DNA segregation and with regulatory restrictions regarding transgenesis3. Therefore, DNA-free delivery has been developed by transfecting preassembled CRISPR–Cas9 ribonucleoproteins into protoplasts4 or in vitro fertilized zygotes5. 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 frequency6,7,8,9. 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.



中文翻译:

使用病毒递送的CRISPR-Cas9进行高效的无DNA植物基因组编辑。

使用CRISPR-Cas核酸酶的基因组编辑技术彻底改变了植物科学,并在作物改良方面具有广阔的前景。常规的转基因介导的CRISPR-Cas试剂递送方法可能与意料之外的基因组变化或损害1,2有关,与涉及外源DNA分离的繁殖周期延长有关转基因的监管限制3。因此,不含DNA的递送已开发通过转染预组装CRISPR-Cas9核糖到原生质体4或体外受精卵5。然而,从这些无壁细胞再生的技术困难使得将这些方法普遍应用于大多数农作物是不切实际的。另外,CRISPR–Cas核糖核蛋白或RNA转录物已被生物弹击入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,尽管频率较低6,7,8,9。在这里,我们报告了一种基于植物负链RNA病毒的载体的工程设计,该载体可在整个CRISPR–Cas9盒的植物体内传递无DNA的植物,从而在模型异源四倍体烟草宿主中实现单个,多重诱变和染色体缺失。从没有被选择的病毒感染组织再生的植物中,超过90%含有定向突变,其中多达57%带有四等位基因可遗传突变。病毒载体甚至在机械传递后仍保持稳定,并且在再生期间或结实后可轻易从突变植物中消除。尽管目标上的活动很多,但目标外的影响(如果有的话)很小。我们的研究为通过病毒感染的植物中CRISPR–Cas9基因编辑提供了一种方便,高效且具有成本效益的方法。

更新日期:2020-06-29
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