Transcriptional perturbation using inactivated CRISPR-nucleases (dCas) is a common method in eukaryotic organisms. While rare examples of dCas9 based tools for prokaryotes have been described, multiplexing approaches are limited due to the used effector nuclease. For the first time, a dCas12a derived tool for the targeted activation and repression of genes was developed. Therefore, a previously described SoxS activator domain was linked to dCas12a to enable programmable activation of gene expression. Proof of principle of transcriptional regulation was demonstrated based on fluorescence reporter assays using the alternative host organism Paenibacillus polymyxa as well as Escherichia coli. Single target and multiplex CRISPR interference targeting the exopolysaccharide biosynthesis of P. polymyxa was shown to emulate polymer compositions of gene knock-outs. Simultaneous expression of 11 gRNAs targeting multiple lactate dehydrogenases and a butanediol dehydrogenase resulted in decreased lactate formation, as well as an increased butanediol production in microaerobic fermentation processes. Even though Cas12a is more restricted in terms of its genomic target sequences compared to Cas9, its ability to efficiently process its own guide RNAs in vivo makes it a promising tool to orchestrate sophisticated genetic reprogramming of bacterial cells or to screen for engineering targets in the genome. The developed tool will accelerate metabolic engineering efforts in the alternative host organism P. polymyxa and might be also applied for other bacterial cell factories.

中文翻译：

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一种基于原核CRISPR-Cas12a的新型工具，可进行可编程的转录激活和抑制

使用灭活的CRISPR核酸酶（dCas）进行转录扰动是真核生物的常见方法。虽然已经描述了用于原核生物的基于dCas9的工具的稀有实例，但由于使用了效应子核酸酶，因此多路复用方法受到限制。首次开发了dCas12a衍生的工具，用于靶向激活和抑制基因。因此，先前描述的SoxS激活域连接到dCas12a，以实现基因表达的可编程激活。转录调节原理的证明是基于荧光报告基因检测，使用了替代宿主生物多粘芽孢杆菌和大肠杆菌。靶向CRISPR胞外多糖生物合成的单靶和多重CRISPR干扰。P. polymyxa被证明可以模拟基因敲除的聚合物组成。11种同时靶向多种乳酸脱氢酶和丁二醇脱氢酶的gRNA的表达导致乳酸形成的减少，以及微需氧发酵过程中丁二醇产量的增加。尽管与Cas9相比，Cas12a在基因组靶序列方面受到更多限制，但其在体内有效处理其自身指导RNA的能力使其成为编排细菌细胞复杂的基因重编程或筛选基因组中工程靶标的有前途的工具。开发的工具将加快替代宿主生物多粘菌的代谢工程工作，也可能会应用于其他细菌细胞工厂。