Pandey Neha, Misra Chitra Seetharam, Rath Devashish
Applied Genomics Section, Bio-Science Group, Bhabha Atomic Research Centre, Mumbai, India.
Life Sciences, Mumbai University, Vidya Nagari, Kalina, Santacruz East, Mumbai, India.
Biochem J. 2025 Jun 11;482(12):BCJ20253056. doi: 10.1042/BCJ20253056.
The structural and mechanistic complexity of Escherichia coli's type I Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated (CRISPR-Cas) system, compared with the multidomain, single effector protein-based type II systems, limits its application in genome editing and silencing. Despite the higher prevalence of the type I endogenous systems in bacteria, significant research has focused on improving the type II systems. While the type-I CRISPR system possesses several advantages over others, it may benefit from further studies to simplify the system for ease of use. To enable this, the dispensability of the type-I Cascade components (Cas8, Cas11, Cas7, Cas5 and Cas6) for genome editing and silencing applications was evaluated in vivo. We created deletion variants of each of the Cascade components and investigated their effects on gene silencing and plasmid interference in two genetically distinct E. coli lineages: BW25113, a K-12 strain that bears an endogenous, albeit repressed type I-E CRISPR system; and BL21, a natural mutant lacking the type I-E CRISPR-Cas system. Cas8, Cas7 and Cas5 were found to be indispensable for gene silencing and plasmid interference. Dispensability of Cas6, which is involved in crRNA maturation, was strain-dependent. Notably, Cas11, which has no definitive function assigned to it, was found to be dispensable for gene silencing and plasmid interference.
与基于多结构域单效应蛋白的II型系统相比，大肠杆菌I型成簇规律间隔短回文重复序列/CRISPR相关（CRISPR-Cas）系统的结构和机制复杂性限制了其在基因组编辑和沉默中的应用。尽管I型内源系统在细菌中更为普遍，但大量研究都集中在改进II型系统上。虽然I型CRISPR系统比其他系统具有一些优势，但进一步研究以简化该系统以便于使用可能会使其受益。为此，在体内评估了I型Cascade组件（Cas8、Cas11、Cas7、Cas5和Cas6）在基因组编辑和沉默应用中的必要性。我们创建了每个Cascade组件的缺失变体，并在两个遗传背景不同的大肠杆菌谱系中研究了它们对基因沉默和质粒干扰的影响：BW25113，一种K-12菌株，带有内源性的I-E型CRISPR系统，尽管该系统受到抑制；以及BL21，一种缺乏I-E型CRISPR-Cas系统的天然突变体。发现Cas8、Cas7和Cas5对于基因沉默和质粒干扰是不可或缺的。参与crRNA成熟的Cas6的必要性因菌株而异。值得注意的是，尚未赋予明确功能的Cas11被发现对于基因沉默和质粒干扰是可有可无的。
