Cas11 augments Cascade functions in type I-E CRISPR system but is redundant for gene silencing and plasmid...

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.