Tobias Bergmiller ^*

• University of Exeter, Exeter, United Kingdom

CRISPR interference (CRISPRi) based on catalytically dead Cas9 nuclease of Streptococcus pyogenes is a programmable and highly flexible tool to interrogate gene function and essentiality in bacteria due to its ability to block transcription elongation at nearly any desired DNA target. Here, I assess how CRISPRi can be programmed to control the life cycle and infectivity of Escherichia coli bacteriophage T7, a highly virulent and obligatory lytic phage, by blocking expression of critical host-dependent promoters and genes that are required for T7 genome translocation and life cycle execution. Here, I focus on the promoters in the non-coding internalisation signal region and T7 RNAP which is controlled by the E. coli-recognised promoter C. Using fluorescent reporters demonstrates that the efficacy of CRISPRi towards promoters in the internalisation signal is limited, and that the phage’s own T7 RNAP can be downregulated very efficiently. Effects on the time to lysis were strongest when the left-most promoter on the leading end of the T7 genome or T7 RNAP was targeted. The stringency of the CRISPRi approach further improved when using multiplex sgRNAs to target multiple phage regions simultaneously, resulting in a 25% increase in the time to lysis and up to 8-fold reduction in plaque size. Overall, this work expands dCas9-dependent CRISPRi as a flexible tool to non-invasively manipulate and probe the lifecycle and infectivity of otherwise native T7 phage.