Interdisciplinary Bioinnovation PhD Program, Tulane University, New Orleans, Louisiana 70118-5665, United States.
Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, Louisiana 70112, United States.
ACS Synth Biol. 2023 Aug 18;12(8):2418-2431. doi: 10.1021/acssynbio.3c00239. Epub 2023 Aug 7.
Phage therapy to treat life-threatening drug-resistant infections has been hampered by technical challenges in phage production. Cell-free bacteriophage synthesis (CFBS) can 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 phages from bench-to-bedside. TXTL harvested from wild type or commonly used bacterial strains was not optimized for bacteriophage production. Here, we demonstrate that TXTL from genetically modified BL21 can be used to enhance phage T7 yields in vitro by CFBS. Expression of 18 BL21 genes was manipulated by inducible CRISPR interference (CRISPRi) mediated by nuclease deficient Cas12a from (dCas12a) 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 translation initiation factor IF-3 () and small RNAs OxyS and CyaR and by repression of RecC subunit exonuclease RecBCD. Continued improvement of CFBS will mitigate phage manufacturing bottlenecks and lower hurdles to widespread adoption of phage therapy.
噬菌体疗法被用于治疗危及生命的耐药性感染，但由于噬菌体生产方面存在技术挑战而受到阻碍。无细胞噬菌体合成（CFBS）可以克服标准噬菌体生产方法的局限性，在体外制造噬菌体病毒粒子。CFBS 利用从细菌裂解物中提取的转录/翻译机制（TXTL）模拟细胞内噬菌体组装，并与试剂结合，合成由噬菌体基因组 DNA 模板编码的蛋白质。这些系统可以实现噬菌体的快速生产和工程化，从而加速噬菌体从实验室到临床的应用。从野生型或常用细菌菌株中提取的 TXTL 并未针对噬菌体生产进行优化。在这里，我们证明了来自遗传修饰的 BL21 的 TXTL 可以通过 CFBS 来提高噬菌体 T7 的体外产量。通过 Cas12a （dCas12a）介导的无核酸酶的 CRISPR 干扰（CRISPRi）来操纵 18 个 BL21 基因的表达，以鉴定与 T7 繁殖相关的基因作为正或负效应因子。显示出显著影响的基因被过表达（正效应因子）或抑制（负效应因子），以修饰用于 CFBS 的 TXTL 的遗传背景。通过过表达翻译起始因子 IF-3（）和小 RNA OxyS 和 CyaR 以及抑制 RecC 亚基外切酶 RecBCD，噬菌体 T7 的 CFBS 产量在体外提高了 10 倍。通过继续改进 CFBS，将减轻噬菌体制造的瓶颈，并降低噬菌体治疗广泛应用的障碍。
