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首页> 外文期刊>Biotechnology and Bioengineering >Developing a Cas9-based tool to engineer native plasmids in Synechocystis sp PCC 6803
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Developing a Cas9-based tool to engineer native plasmids in Synechocystis sp PCC 6803

机译：在SyneChocystis SP PCC 6803中开发基于Cas9的工具。在SyneChocystis SP PCC 6803中工程

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The oxygenic photosynthetic bacterium Synechocystis sp. PCC 6803 (S6803) is a model cyanobacterium widely used for fundamental research and biotechnology applications. Due to its polyploidy, existing methods for genome engineering of S6803 require multiple rounds of selection to modify all genome copies, which is time-consuming and inefficient. In this study, we engineered the Cas9 tool for one-step, segregation-free genome engineering. We further used our Cas9 tool to delete three of seven S6803 native plasmids. Our results show that all three small-size native plasmids, but not the large-size native plasmids, can be deleted with this tool. To further facilitate heterologous gene expression in S6803, a shuttle vector based on the native plasmid pCC5.2 was created. The shuttle vector can be introduced into Cas9-containing S6803 in one step without requiring segregation and can be stably maintained without antibiotic pressure for at least 30 days. Moreover, genes encoded on the shuttle vector remain functional after 30 days of continuous cultivation without selective pressure. Thus, this study provides a set of new tools for rapid modification of the S6803 genome and for stable expression of heterologous genes, potentially facilitating both fundamental research and biotechnology applications using S6803.
机译：含氧光合细菌综合症Sp。 PCC 6803（S6803）是一种模型的蓝藻，广泛用于基本研究和生物技术应用。由于其多倍体，S6803的基因组工程现有方法需要多轮选择来修饰所有基​​因组拷贝，这是耗时和效率的效率。在这项研究中，我们为一步进行了一步的自由基因组工程设计了Cas9工具。我们进一步使用了CAS9工具来删除七个S6803本地质粒中的三种。我们的研究结果表明，所有三种小型本地质粒，但不是大尺寸的天然质粒，可以用这个工具删除。为了进一步促进S6803中的异源基因表达，产生基于天然质粒PCC5.2的梭子载体。在不需要偏析的情况下，可以在一步中引入含Cas9的S6803的梭矢量，并且可以在没有抗生素的情况下稳定地保持至少30天。此外，在连续培养的30天后，在梭子载体上编码的基因在没有选择性压力的情况下在连续培养后保持官能。因此，本研究提供了一组新工具，用于快速修饰S6803基因组，并且用于使用S6803的稳定表达异源基因的表达，促进基本研究和生物技术应用。

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