Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation
Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation
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DOI：
10.1007/s10142-019-00665-4
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发表时间：
2020-01-01
影响因子：
2.9
通讯作者：
Stuttmann, Johannes
中科院分区：
文献类型：
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作者：
Ordon, Jana;Bressan, Mauro;Stuttmann, Johannes
模式植物拟南芥属的遗传资源包括参考登录号哥伦比亚中几乎任何单个基因缺陷的突变株系。然而，基因冗余和/或紧密连锁通常使得从分离群体中分离缺乏特定功能或基因组的所需品系极其费力或甚至不可能。因此，我们在此评估了通过基于Cas9的核酸酶和多重化来灭活多个基因的策略和效率。在第一次尝试中，我们成功地分离出携带70 kb缺失的突变株系，该突变株系通过基于PCR的筛选许多个体，在T-2代中以类似于1.6%的频率发生。然而，我们未能分离出缺乏Lhcb 1基因的品系，Lhcb 1基因存在于拟南芥基因组中的两个位点上的五个拷贝中。为了提高我们的基于Cas9的核酸酶系统的效率，系统地比较了控制Cas9表达水平和时机的调控序列。事实上，与先前的泛素启动子相比，使用DD 45和RPS 5a启动子将我们的基因组编辑系统的效率提高了约25-30倍。使用具有RPS 5a启动子驱动的Cas9的优化的基因组编辑系统，缺乏可检测量的Lhcb 1蛋白的puplatin五元组突变体系代表约30%的T-I转化体。这些结果显示了改进的基因组编辑系统如何促进复杂突变等位基因的分离，这些等位基因以前被认为是不可能产生的，即使在单个（T-1）世代中也是如此。
Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of similar to 1.6% in the T-2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T-1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T-1) generation.