Geminivirus-Mediated Genome Editing in Potato (Solanum tuberosum L.) Using Sequence-Specific Nucleases.
Geminivirus-Mediated Genome Editing in Potato (Solanum tuberosum L.) Using Sequence-Specific Nucleases.
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DOI：
10.3389/fpls.2016.01045
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发表时间：
2016
影响因子：
5.6
通讯作者：
Douches DS
中科院分区：
文献类型：
--
作者：
Butler NM;Baltes NJ;Voytas DF;Douches DS
利用序列特异性核酸酶(SSN)进行基因组编辑正在迅速发展，用于作物物种的基因工程。利用锌指核酸酶(ZFN)、转录激活物样效应核酸酶(TALEN)和成簇的规则间隔的短回文重复/CRISPR相关系统(CRISPR/CA)来诱导双链断裂，几乎可以靶向任何序列进行修饰。通过非同源末端连接的定向突变(NHEJ)已被广泛地证明是植物DNA修复的首选途径。然而，通过同源重组(HR)的基因打靶仍然难以捉摸，但可能是一种强大的定向DNA修复工具。为了克服与基因打靶相关的障碍，双生病毒复制子(GVR)被用来传递针对马铃薯乙酰乳酸SYNTHase 1(ALS1)基因的SSN和修复模板，该模板旨在将除草剂抑制点突变整合到ALS1基因座中。用GVRS修饰的转化事件持有能够支持降低除草剂敏感性表型的点突变，而用传统T-DNA转化的事件没有检测到突变，与野生型相似。转化事件的再生改善了对支持更强的降低除草剂敏感性表型的点突变的检测。这些结果证明了利用双生病毒在植物物种中传递基因组编辑试剂，以及在营养繁殖物种中进行基因靶向的一种新方法。
Genome editing using sequence-specific nucleases (SSNs) is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas) for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via non-homologous end-joining (NHEJ) has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination (HR) remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR) was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and a novel approach to gene targeting in a vegetatively propagated species.