Genomic Sciences Graduate Program, North Carolina State University, Raleigh, NC 27695.
Department of Food, Bioprocessing & Nutrition Sciences, North Carolina State University, Raleigh, NC 27606.
Proc Natl Acad Sci U S A. 2022 Nov 16;119(46):e2206744119. doi: 10.1073/pnas.2206744119. Epub 2022 Nov 7.
Investigation of microbial gene function is essential to the elucidation of ecological roles and complex genetic interactions that take place in microbial communities. While microbiome studies have increased in prevalence, the lack of viable in situ editing strategies impedes experimental progress, rendering genetic knowledge and manipulation of microbial communities largely inaccessible. Here, we demonstrate the utility of phage-delivered CRISPR-Cas payloads to perform targeted genetic manipulation within a community context, deploying a fabricated ecosystem (EcoFAB) as an analog for the soil microbiome. First, we detail the engineering of two classical phages for community editing using recombination to replace nonessential genes through Cas9-based selection. We show efficient engineering of T7, then demonstrate the expression of antibiotic resistance and fluorescent genes from an engineered λ prophage within an host. Next, we modify λ to express an APOBEC-1-based cytosine base editor (CBE), which we leverage to perform C-to-T point mutations guided by a modified Cas9 containing only a single active nucleolytic domain (nCas9). We strategically introduce these base substitutions to create premature stop codons in-frame, inactivating both chromosomal () and plasmid-encoded genes (mCherry and ampicillin resistance) without perturbation of the surrounding genomic regions. Furthermore, using a multigenera synthetic soil community, we employ phage-assisted base editing to induce host-specific phenotypic alterations in a community context both in vitro and within the EcoFAB, observing editing efficiencies from 10 to 28% across the bacterial population. The concurrent use of a synthetic microbial community, soil matrix, and EcoFAB device provides a controlled and reproducible model to more closely approximate in situ editing of the soil microbiome.
研究微生物基因功能对于阐明微生物群落中发生的生态作用和复杂遗传相互作用至关重要。虽然微生物组研究的普及度有所增加，但缺乏可行的原位编辑策略阻碍了实验进展，使得微生物群落的遗传知识和操作在很大程度上无法实现。在这里，我们展示了噬菌体递送的 CRISPR-Cas 有效负载在群落背景下进行靶向遗传操作的效用，将人工生态系统（EcoFAB）作为土壤微生物组的模拟物。首先，我们详细介绍了两种经典噬菌体的工程改造，通过 Cas9 为基础的选择利用重组来取代非必需基因。我们展示了 T7 的高效工程改造，然后证明了来自工程化 λ 噬菌体前噬菌体的抗生素抗性和荧光基因在宿主内的表达。接下来，我们修饰了 λ 噬菌体以表达基于 APOBEC-1 的胞嘧啶碱基编辑器（CBE），我们利用它来指导经修饰的 Cas9 进行 C 到 T 的点突变，该 Cas9 仅包含单个活性核酸酶结构域（nCas9）。我们策略性地引入这些碱基替换，在框架内产生过早的终止密码子，使染色体（）和质粒编码基因（mCherry 和氨苄青霉素抗性）失活，而周围基因组区域不受干扰。此外，我们使用多代合成土壤群落，通过噬菌体辅助的碱基编辑在体外和 EcoFAB 内的群落背景下诱导宿主特异性表型改变，在整个细菌群体中观察到 10%至 28%的编辑效率。同时使用合成微生物群落、土壤基质和 EcoFAB 设备提供了一个可控且可重复的模型，更接近原位编辑土壤微生物组。
