A CRISPRi-dCas9 System for Archaea and Its Use To Examine Gene Function during Nitrogen Fixation by Methanosarcina acetivorans
A CRISPRi-dCas9 System for Archaea and Its Use To Examine Gene Function during Nitrogen Fixation by Methanosarcina acetivorans
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DOI：
10.1128/aem.01402-20
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发表时间：
2020-11-01
影响因子：
4.4
通讯作者：
Lessner, Daniel J.
中科院分区：
文献类型：
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作者：
Dhamad, Ahmed E.;Lessner, Daniel J.
基于CRISPR的系统正在成为操纵许多细胞过程的首要方法。在这项研究中，开发了一种简单有效的CRISPR干扰（CRISPRi）系统，用于古细菌中的靶向基因阻遏。通过用催化死亡的Cas9（dCas 9）替换Cas9来重新利用嗜乙酸甲烷八叠球菌CRISPR-Cas9系统，以产生用于靶向基因阻遏的CRISPRi-dCas 9系统。为了测试该系统的效用，靶向参与氮（N-2）固定的基因用于dCas 9介导的阻遏。首先，编码钼固氮酶的nif操纵子（nifHI（1）I（2）DKEN）被单独的指导RNA（gRNA）靶向，一个靶向启动子，另一个靶向nifD。值得注意的是，M.通过dCas 9介导的用每种gRNA对nif操纵子的阻遏，消除了食乙酸菌与N-2的结合。在表达gRNA的两种菌株中，nif转录物的丰度降低>90%，并且在细胞裂解物中未检测到NifD。接下来，我们针对固氮酶辅因子生物合成所需的NifB。靶向NifB编码序列的gRNA的表达使nifB转录物丰度降低>85%，并且损害但不消除M.醋食菌素与N2。最后，为了确定使用CRISPRi-dCas 9研究基因调控的能力，靶向了编码nif操纵子阻遏物亚基的nrpR 1。nrpR 1阻遏菌株与N-2正常生长，但nif操纵子转录丰度增加，与NrpR 1作为阻遏物一致。这些结果突出了该系统的实用性，由此当用dCas 9表达时，单个gRNA可以阻断M中靶基因和操纵子的转录。重要性基因工具需要了解和操纵古细菌的生物学，在生物圈中发挥关键作用。产甲烷古菌（产甲烷菌）是生物生产甲烷所必需的，甲烷是全球碳循环的中间体，是一种重要的温室气体，也是一种生物燃料。据我们所知，模型产甲烷菌Methanosarcina acetivorans中的CRISPRi-dCas 9系统是古细菌中第一个基于Cas9的CRISPR干扰系统。结果表明，该系统是非常有效的靶向基因阻遏，并提供了新的见解甲烷菌，唯一的古细菌固氮。总的来说，CRISPRi-dCas 9系统提供了一种简单但强大的遗传工具来控制产甲烷菌中靶基因和操纵子的表达。
CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N-2) fixation were targeted for dCas9-mediated repression. First, the nif operon (nifHI(1)I(2)DKEN) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD. Remarkably, growth of M. acetivorans with N-2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N2. Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1, encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N-2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivorans.IMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas, and a biofuel. The CRISPRi-dCas9 system in the model methanogen Methanosarcina acetivorans is, to our knowledge, the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens.