Ban, H.; Rondthaler, S. N.; Lebovich, M.; Lora, M. A.; Ugbesia, B.; Andrews, L. B.

CRISPR interference (CRISPRi) has emerged as a versatile approach for targeted gene repression in many organisms, including microbes and bacteria, due to the simple design of sequence-specific transcriptional silencing of gene expression. However, the strain-specific effects on repression efficiency and the host when translating a CRISPRi system from a laboratory strain to non-model strains are not well understood, yet they can present important limitations to its use. Here, we investigated the repression efficiency and toxicity of three CRISPRi systems (one dCas9 and two dCas12a variants) across four different Escherichia coli strains, including a laboratory K-12 strain (MG1655) and three non-model strains that are clinical isolates (probiotic Nissle 1917, uropathogenic CFT073, and uropathogenic UMN026). We evaluated the repression in each strain using sets of guide RNAs (gRNAs) targeting along the gene sequence and assayed cytotoxicity of expressing each dCas protein. Growth toxicity from expression of the different dCas proteins notably differed and showed high variation between some host strains. We also observed variable repression among the strains and notably poorer repression in multiple clinical strains. Therefore, we developed a dual gRNA CRISPRi system for enhanced gene silencing among the strains, which achieved up to 824-fold repression in CFT073. The results demonstrate that strain-specific design considerations can arise when a CRISPRi genetic system is transferred to a closely related bacterial strain. These findings provide insight into the relationships between criteria used for CRISPRi genetic design and in vivo activity across non-model E. coli strains, providing guidelines for diverse applications of these tools.