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在Clostridium beijerinckii NCIMB 8052中使用CRISPR-Cas12a进行多重基因组工程

Constantinos Patinios^1,2, Stijn T de Vries¹, Mamou Diallo^1,3

¹Laboratory of Microbiology, Wageningen University and Research, Stippeneng 4, 6708 WE, Wageningen, The Netherlands.

Scientific reports

|June 22, 2023

概括

研究人员开发了一种新的CRISPR-Cas12a基因组工程工具,用于Clostridium beijerinckii,可实现高效的单基因和多基因淘汰,以改善工业酸盐-butanol-乙醇生产.

科学领域:

合成生物学合成生物学
微生物生物技术微生物生物技术
基因组工程是基因组工程.

背景情况:

克洛斯特里物种对于工业乙烯-布坦醇-乙醇 (ABE) 生产至关重要.
发酵和基因组工程的进步正在推动Clostridium在生物技术中的重新出现.
克里斯普尔-卡斯系统是微生物基因组工程的强大工具.

研究的目的:

为了扩大克里斯普尔-卡斯工具箱的Clostridium beijerinckii.
在Clostridium beijerinckii中开发和描述一个CRISPR-Cas12a基因组工程系统NCIMB 8052.2.
为了证明这个系统对单个和多重基因淘汰的实用性.

主要方法:

使用FnCas12a基因开发一个CRISPR-Cas12a系统.
通过可诱导酶的促进剂控制FnCas12a的表达.
针对C. beijerinckii的特定基因的引导RNA的设计 NCIMB 8052.

主要成果:

在五个目标基因 (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832) 中实现了高效的 (25-100%) 单基因淘汰.
通过与18%的效率同时淘汰spo0A和upp基因,证明了成功的多重基因工程.
确定了在CRISPR数组中的间隔器序列和位置会影响编辑效率.

结论:

开发的CRISPR-Cas12a工具是有效的基因组工程在Clostridium beijerinckii.
该系统可促进单基因和多基因基因编辑,从而提升代谢工程能力.
优化CRISPR阵列设计对于最大限度地提高C. beijerinckii的编辑效率至关重要.

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