Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans
Cas9-mediated genome editing in the methanogenic archaeon Methanosarcina acetivorans
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DOI：
10.1073/pnas.1618596114
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发表时间：
2017-03-14
影响因子：
11.1
通讯作者：
Metcalf, William W.
中科院分区：
文献类型：
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作者：
Nayak, Dipti D.;Metcalf, William W.
虽然Cas9介导的基因组编辑已被证明是真核生物中一种强大的遗传工具，但由于其在细菌中的低效靶向或修复，其在细菌中的应用一直受到限制；其在古生物中的应用尚未见报道。在这里，我们描述了一种由Cas9介导的基因组编辑工具的开发，该工具允许对生长缓慢的产甲烷始祖菌乙酸甲烷单胞菌进行便捷的遗传操作。通过同源定向修复引入插入和缺失都是非常有效和精确的，相对于转化效率，发生频率约为20%，基本上在所有被检查的转化子中都发现了所需的突变。没有观察到偏离目标的活动。我们还观察到，多个单引导RNA可以在同一转录本中表达，从而减小了突变质粒的大小，同时简化了它们的设计。Cas9介导的基因组编辑将构建突变体所需的时间减少了一半以上(3wk比8wk)，并允许高效率地同时构建双突变体，以指数级减少复杂菌株构建所需的时间。此外，共表达来自密切相关的考古子沼泽甲烷菌的非同源末端连接(NHEJ)机制，允许高效的Cas9介导的基因组编辑而不需要修复模板。依赖于NHEJ的突变包括长度从75到2.7kb的缺失，其中大部分似乎发生在自然发生的微同源区域。同源导向的修复依赖和NHEJ依赖的基因组编辑工具的组合构成了一个强大的遗传系统，该系统能够方便地插入和删除基因，合理修改基因表达，并测试基因的重要性。
Although Cas9-mediated genome editing has proven to be a powerful genetic tool in eukaryotes, its application in Bacteria has been limited because of inefficient targeting or repair; and its application to Archaea has yet to be reported. Here we describe the development of a Cas9-mediated genome-editing tool that allows facile genetic manipulation of the slow-growing methano-genic archaeon Methanosarcina acetivorans. Introduction of both insertions and deletions by homology-directed repair was remarkably efficient and precise, occurring at a frequency of approximately 20% relative to the transformation efficiency, with the desired mutation being found in essentially all transformants examined. Off-target activity was not observed. We also observed that multiple single-guide RNAs could be expressed in the same transcript, reducing the size of mutagenic plasmids and simultaneously simplifying their design. Cas9-mediated genome editing reduces the time needed to construct mutants by more than half (3 vs. 8 wk) and allows simultaneous construction of double mutants with high efficiency, exponentially decreasing the time needed for complex strain constructions. Furthermore, coexpression the non-homologous end-joining (NHEJ) machinery from the closely related archaeon, Methanocella paludicola, allowed efficient Cas9-mediated genome editing without the need for a repair template. The NHEJ-dependent mutations included deletions ranging from 75 to 2.7 kb in length, most of which appear to have occurred at regions of naturally occurring microhomology. The combination of homology-directed repair-dependent and NHEJ-dependent genome-editing tools comprises a powerful genetic system that enables facile insertion and deletion of genes, rational modification of gene expression, and testing of gene essentiality.