Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

Phage therapy totreat life-threatening drug-resistant infectionshas been hampered by technical challenges in phage production. Cell-freebacteriophage synthesis (CFBS) can overcome the limitations of standardphage production methods by manufacturing phage virions in vitro.CFBS mimics intracellular phage assembly using transcription/translationmachinery (TXTL) harvested from bacterial lysates and combined withreagents to synthesize proteins encoded by a phage genomic DNA template.These systems may enable rapid phage production and engineering toaccelerate phages from bench-to-bedside. TXTL harvested from wildtype or commonly used bacterial strains was not optimized for bacteriophageproduction. Here, we demonstrate that TXTL from genetically modified E. coli BL21 can be used to enhance phage T7 yieldsin vitro by CFBS. Expression of 18 E. coli BL21 genes was manipulated by inducible CRISPR interference (CRISPRi)mediated by nuclease deficient Cas12a from F. novicida (dFnCas12a) to identify genes implicated in T7propagation as positive or negative effectors. Genes shown to havea significant effect were overexpressed (positive effectors) or repressed(negative effectors) to modify the genetic background of TXTL harvestedfor CFBS. Phage T7 CFBS yields were improved by up to 10-fold in vitrothrough overexpression of translation initiation factor IF-3 (infC) and small RNAs OxyS and CyaR and by repression ofRecC subunit exonuclease RecBCD. Continued improvement of CFBS willmitigate phage manufacturing bottlenecks and lower hurdles to widespreadadoption of phage therapy.