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重新利用内源性II型CRISPR-Cas9系统用于Streptococcus thermophilus中的基因组编辑

Shuai Ma^1,2, Feiyu Wang¹, Zhang Xuejing¹

¹State Key Laboratory of Microbial Technology, Shandong University, Qingdao, People's Republic of China.

Biotechnology and bioengineering

|November 23, 2023

概括

我们在Streptococcus thermophilus中重新应用了CRISPR系统,以实现精确的基因组编辑. 这个工具包使得高效的基因操纵能够在乳制品发酵中得到增强的生物技术应用.

科学领域:

微生物学微生物学
分子生物学分子生物学
生物技术是生物技术.

背景情况:

热菌 (Streptococcus thermophilus) 对于工业乳制品发酵至关重要.
有限的基因操纵工具阻碍了它的生物技术潜力.

研究的目的:

重新利用内生CRISPR1和CRISPR3系统进行精确的基因组工程在S. thermophilus.
开发一种工具包,以有效地对S. thermophilus进行基因操纵.

主要方法:

重新利用内源型II-ACRISPR-Cas9系统 (CRISPR1和CRISPR3) 的目的.
在S. thermophilus LMD-9.9中提供具有DNA修复模板的自定位CRISPR阵列.
向基因包括lacZ和eps基因集群.

主要成果:

使用CRISPR3与1.5kb修复模板实现了高效的基因编辑 (在lacZ中删除高达90%).
以75%-100%的效率产生各种编辑结果 (停止编码子插入,单核酸变异,DNA片段插入).
在eps基因集群中创建了六个单基因淘汰突变 (29%-80%的效率),确定了epsA,epsE和epsG作为外聚糖生物合成的关键.

结论:

重用本地II-A型CRISPR-Cas9系统为S. thermophilus的精确基因组工程提供了一个多功能工具包.
这种方法显著提高了S. thermophilus在生物技术应用中的潜力.
确定了调节外聚糖生物合成的关键基因,为菌株改进提供了目标.

关键词:

在CRIPSR-Cas9中使用.克里斯普尔1是指克里斯普尔.克里斯普尔3是指克里斯普尔.S. 热的热的热的这是内源性的.基因组编辑基因组编辑

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