The use of molecular tools based on the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems has rapidly advanced genetic engineering. These molecular biological tools have been applied for different genetic engineering purposes in multiple organisms, including the quite rarely explored Paenibacillus polymyxa. However, only limited studies on large cluster deletion and multiplex genome editing have been described for this highly interesting and versatile bacterium. Here, we demonstrate the utilization of a Cas9-based system to realize targeted deletions of four biosynthetic gene clusters in the range of 12 kb to 41 kb by the use of a single targeting sgRNA. Furthermore, we also harness the system for multiplex editing of genes and large genomic regions. Multiplex deletion was achieved with more than 80 % efficiency, while simultaneous integration at two distantly located sites was obtained with 58 % efficiency. The findings reported in this study are anticipated to accelerate future research in P. polymyxa and related species.

中文翻译：

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CRISPR-Cas9 介导的多粘类芽孢杆菌的大簇删除和多重基因组编辑

基于成簇的规则间隔短回文重复序列-Cas (CRISPR-Cas) 系统的分子工具的使用迅速推进了基因工程。这些分子生物学工具已在多种生物体中用于不同的基因工程目的，包括很少探索的多粘类芽孢杆菌. 然而，对于这种非常有趣且用途广泛的细菌，仅描述了对大簇缺失和多重基因组编辑的有限研究。在这里，我们展示了利用基于 Cas9 的系统通过使用单个靶向 sgRNA 实现 12 kb 至 41 kb 范围内四个生物合成基因簇的靶向缺失。此外，我们还利用该系统对基因和大基因组区域进行多重编辑。以超过 80% 的效率实现多重删除，同时以 58% 的效率在两个距离较远的位点进行整合。本研究报告的结果预计将加速对多粘假单胞菌和相关物种的未来研究。