CRISPR/Cas precision: do we need to worry about off-targeting in plants?
CRISPR/Cas precision: do we need to worry about off-targeting in plants?
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DOI：
10.1007/s00299-018-2355-9
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发表时间：
2019-04-01
影响因子：
6.2
通讯作者：
Nekrasov, Vladimir
中科院分区：
文献类型：
--
作者：
Hahn, Florian;Nekrasov, Vladimir
CRISPR/Cas技术最近已成为植物及其他植物靶向基因组修饰的首选工具。尽管CRSIPR/Cas提供了在感兴趣的基因组基因座处引入变化的快速且简便的方式，但其应用与脱靶相关，即在基因组内的脱靶位点处引入非预期突变，这在哺乳动物领域中已被频繁报道。在这里，我们总结了目前关于CRISPR/Cas在植物系统中的精确性的知识，并提供了避免植物中脱靶突变以及意外的靶向变化的最新策略的总结。这些包括使用天然（例如Cas 12 a）或工程化（例如SpCas 9-HF）CRISPR/Cas核酸酶，其特征在于与常用的野生型SpCas 9相比具有更高的精确度。此外，我们还讨论了核糖核蛋白（RNP）形式的CRISPR/Cas核酸酶的使用，作为减少植物脱靶的一种选择。最后，我们得出结论，减少CRISPR/Cas脱靶的最重要因素仍然是仔细选择靶序列，为此，我们提供了可用的在线软件工具和实验指导的概述。
The CRISPR/Cas technology has recently become the tool of choice for targeted genome modification in plants and beyond. Although CRSIPR/Cas offers a rapid and facile way of introducing changes at genomic loci of interest, its application is associated with off-targeting, i.e. introduction of unintended mutations at off-target sites within the genome, which has been reported frequently in the mammalian field. Here we summarise the current knowledge on the precision of CRISPR/Cas in plant systems and provide a summary of state-of the-art strategies for avoiding off-target mutations, as well as unintended on-target changes, in plants. These include using natural (e.g. Cas12a) or engineered (e.g. SpCas9-HF) CRISPR/Cas nucleases characterised by higher precision, as compared to the commonly used wild type SpCas9. In addition, we discuss the usage of CRISPR/Cas nucleases in the form of ribonucleoproteins (RNPs) as an option for reducing off-targeting in plants. Finally, we conclude that the most important factor for reducing CRISPR/Cas off-targeting remains careful selection of target sequences, for which we provide an overview of available online software tools and experimental guidance.