(623f) Cell Free Bacteriophage Synthesis Via Engineered Strains to Improve Yield

Cell-free bacteriophage synthesis (CFBS) is an approach to overcome the limitations of standard phage production methods by manufacturing phage virions in vitro. CFBS mimics intracellular phage assembly using transcription/translation machinery (TXTL) harvested from bacterial lysates and combined with reagents to synthesize proteins encoded by a phage genomic DNA template. These systems may enable rapid phage production and engineering to accelerate phage from bench-to-bedside. TXTL harvested from wild type or commonly used strains are not optimized for bacteriophage production, however. In this presentation, we will demonstrate that TXTL from genetically modified E. coli BL21 can be used to enhance phage T7 yields in 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 T7 propagation as positive or negative effectors. Genes shown to have a significant effect were overexpressed (positive effectors) or repressed (negative effectors) to modify the genetic background of TXTL harvested for CFBS. Phage T7 CFBS yields were improved by up to 10-fold in vitro through overexpression of genes associated with translation, regulation, and exonuclease activity. We also discuss efforts to use this CFBS platform for rapid bacteriophage genome engineering with implications for improved phage therapy. Continued improvement of CFBS will mitigate phage manufacturing bottlenecks and lower hurdles to widespread adoption of phage therapy.