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在Lacticaseibacillus paracasei中,应用SpCas9抑制剂BRD0539用于基于CRISPR/Cas9的遗传工具

Mizuna Tamaki¹, Tsutomu Chiku¹, Shunya Suzuki²

¹Department of Agricultural Chemistry, Graduate School of Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan.

Bioscience of microbiota, food and health

|January 7, 2025

概括

一种新的小分子抑制剂BRD0539部分抑制了Streptococcus pyogenes Cas9 (SpCas9) 在Lacticaseibacillus paracasei中的活性,用于基因编辑和CRISPR干扰应用.

科学领域:

微生物学微生物学
分子生物学分子生物学
基因工程是一种基因工程.

背景情况:

聚类正规间隔短时间的Palindromic重复 (CRISPR) -Cas系统是各种生物体基因组编辑的强大工具.
对于细菌应用,特别是乳酸细菌,需要改进.
控制CRISPR-Cas9核酶活性对于精确的基因修饰至关重要.

研究的目的:

为了研究BRD0539,一个小分子抑制剂,在调节Lacticaseibacillus paracasei中的CRISPR-Cas9活性中的有效性.
在L. paracasei.中开发和验证CRISPR-SpCas9系统用于基因编辑和CRISPR干扰 (CRISPRi).
评估抑制剂在这种细菌物种中调节CRISPRi中的作用.

主要方法:

开发并验证了一种针对L. paracasei. sirA基因的CRISPR-SpCas9系统.
利用BRD0539在L. paracasei和Escherichia coli的体内DNA裂变过程中抑制SpCas9的活性.
在L. paracasei中构建了一个CRISPRi系统,使用一个不活跃的SpCas9和一个针对sirA基因的特定单向导RNA (sgRNA).

主要成果:

在大肠杆菌和L. paracasei中,BRD0539证明了SpCas9介导的DNA裂变的部分抑制.
在L. paracasei.中使用CRISPRi系统成功实现了sirA的基因沉默.
发现CRISPRi活性取决于抑制剂的存在,当BRD0539缺席时被废除.

结论:

在L. paracasei.中,BRD0539显示出作为调节CRISPR-SpCas9活动的工具的潜力.
开发的CRISPRi系统为乳酸细菌的基因表达调节提供了可控制的方法.
这些发现有助于扩大微生物研究的CRISPR工具箱,特别是乳酸细菌.

关键词:

BRD053939 这是一个很好的例子.这就是CRISPR/Cas9的作用.克里斯普里是指克里斯普里.乳糖酶乳糖酶乳糖酶乳糖酶乳糖酶乳糖酶乳糖酶乳糖酶乳糖酶乳糖酶乳糖酶乳糖酶乳酸细菌是一种乳酸细菌.遗传修饰是一种基因改造.

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