Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco
Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco
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DOI：
10.1007/s11103-019-00907-w
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发表时间：
2019-11-01
影响因子：
5.1
通讯作者：
Lin, Choun-Sea
中科院分区：
文献类型：
--
作者：
Hsu, Chen-Tran;Cheng, Yu-Jung;Lin, Choun-Sea
原生质体可以单独或同时使用几种不同的CRISPR系统进行基因组编辑，并且可以在再生的非嵌合植物中回收所得突变。原生质体转染和再生系统是用于CRISPR/Cas诱变和基因组编辑的有用平台。在这项研究中，我们证明了使用Cpf 1（Cas 12 a）和nCas 9激活诱导的胞苷脱氨酶（nCas 9-Target-AID）系统诱变烟草原生质体并再生携带所得突变的植物。我们分析了Cas 12 a介导的八氢番茄红素去饱和酶（PDS）诱变的再生体以及来自野生型原生质体的再生体的20个后代植物，并证实它们的基因型以孟德尔方式遗传。我们使用Cas9切口酶（nCas 9）-胞苷脱氨酶对烟草原生质体中的乙烯受体1（ETR 1）基因进行C至T编辑，并获得编辑的再生体。当编辑效率低时，很难获得多倍体基因组的纯合编辑。部分编辑的再生体的第二轮诱变（两步转染方案）允许我们在不需要有性繁殖的情况下获得ETR 1完全编辑的再生体。我们使用一步或两步转染平台应用三种不同的Cas系统（SaCas 9、Cas 12 a和nCas 9-Target AID）以获得三重突变和/或编辑的烟草再生体。我们的研究结果表明，这三个Cas系统可以在单个细胞内同时发挥作用。
Key message Protoplasts can be used for genome editing using several different CRISPR systems, either separately or simultaneously, and that the resulting mutations can be recovered in regenerated non-chimaeric plants. Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this study, we demonstrate the use of Cpf1 (Cas12a) and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID) systems to mutagenize Nicotiana tabacum protoplasts and to regenerate plants harboring the resulting mutations. We analyzed 20 progeny plants of Cas12a-mediated phytoene desaturase (PDS) mutagenized regenerants, as well as regenerants from wild-type protoplasts, and confirmed that their genotypes were inherited in a Mendelian manner. We used a Cas9 nickase (nCas9)-cytidine deaminase to conduct C to T editing of the Ethylene receptor 1 (ETR1) gene in tobacco protoplasts and obtained edited regenerates. It is difficult to obtain homozygous edits of polyploid genomes when the editing efficiency is low. A second round of mutagenesis of partially edited regenerants (a two-step transfection protocol) allowed us to derive ETR1 fully edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) using either a one-step or a two-step transfection platform to obtain triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.