Pub Date : 2024-10-22DOI: 10.1021/acssynbio.4c00183
Crystal Chen, Lei S Qi
Manipulating RNA species in mammalian cells has emerged as an important strategy for precise gene expression control. Here we review recent advances in precision transcriptome editing with a focus on tools that engineer specific transcripts for abundance, translation, base editing, alternative isoforms, and chemical modifications. While some of these methods have demonstrated efficiency in therapeutically relevant cellular or in vivo models, most require further study on their clinical safety and efficacy. Precision transcriptome engineering holds great potential for both mechanistic study of RNA biology and future gene and cell-based therapeutic applications.
{"title":"Precision Transcriptome Editing.","authors":"Crystal Chen, Lei S Qi","doi":"10.1021/acssynbio.4c00183","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00183","url":null,"abstract":"<p><p>Manipulating RNA species in mammalian cells has emerged as an important strategy for precise gene expression control. Here we review recent advances in precision transcriptome editing with a focus on tools that engineer specific transcripts for abundance, translation, base editing, alternative isoforms, and chemical modifications. While some of these methods have demonstrated efficiency in therapeutically relevant cellular or <i>in vivo</i> models, most require further study on their clinical safety and efficacy. Precision transcriptome engineering holds great potential for both mechanistic study of RNA biology and future gene and cell-based therapeutic applications.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142453379","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-19DOI: 10.1021/acssynbio.4c00534
Urte Tomasiunaite, Tess Brewer, Korinna Burdack, Sophie Brameyer, Kirsten Jung
Protein synthesis is influenced by the chemical and structural properties of the amino acids incorporated into the polypeptide chain. Motifs containing consecutive prolines can slow the translation speed and cause ribosome stalling. Translation elongation factor P (EF-P) facilitates peptide bond formation in these motifs, thereby alleviating stalled ribosomes and restoring the regular translational speed. Ribosome pausing at various polyproline motifs has been intensively studied using a range of sophisticated techniques, including ribosome profiling, proteomics, and in vivo screening, with reporters incorporated into the chromosome. However, the full spectrum of motifs that cause translational pausing in Escherichia coli has not yet been identified. Here, we describe a plasmid-based dual reporter for rapid assessment of pausing motifs. This reporter contains two coupled genes encoding mScarlet-I and chloramphenicol acetyltransferase to screen motif libraries based on both bacterial fluorescence and survival. In combination with a diprolyl motif library, we used this reporter to reveal motifs of different pausing strengths in an E. coli strain lacking efp. Subsequently, we used the reporter for a high-throughput screen of four motif libraries, with and without prolines at different positions, sorted by fluorescence-associated cell sorting (FACS) and identify new motifs that influence the translational efficiency of the fluorophore. Our study provides an in vivo platform for rapid screening of amino acid motifs that affect translational efficiencies.
{"title":"Versatile Dual Reporter to Identify Ribosome Pausing Motifs Alleviated by Translation Elongation Factor P.","authors":"Urte Tomasiunaite, Tess Brewer, Korinna Burdack, Sophie Brameyer, Kirsten Jung","doi":"10.1021/acssynbio.4c00534","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00534","url":null,"abstract":"<p><p>Protein synthesis is influenced by the chemical and structural properties of the amino acids incorporated into the polypeptide chain. Motifs containing consecutive prolines can slow the translation speed and cause ribosome stalling. Translation elongation factor P (EF-P) facilitates peptide bond formation in these motifs, thereby alleviating stalled ribosomes and restoring the regular translational speed. Ribosome pausing at various polyproline motifs has been intensively studied using a range of sophisticated techniques, including ribosome profiling, proteomics, and in vivo screening, with reporters incorporated into the chromosome. However, the full spectrum of motifs that cause translational pausing in <i>Escherichia coli</i> has not yet been identified. Here, we describe a plasmid-based dual reporter for rapid assessment of pausing motifs. This reporter contains two coupled genes encoding mScarlet-I and chloramphenicol acetyltransferase to screen motif libraries based on both bacterial fluorescence and survival. In combination with a diprolyl motif library, we used this reporter to reveal motifs of different pausing strengths in an <i>E. coli</i> strain lacking <i>efp</i>. Subsequently, we used the reporter for a high-throughput screen of four motif libraries, with and without prolines at different positions, sorted by fluorescence-associated cell sorting (FACS) and identify new motifs that influence the translational efficiency of the fluorophore. Our study provides an in vivo platform for rapid screening of amino acid motifs that affect translational efficiencies.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142453381","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-19DOI: 10.1021/acssynbio.4c00337
Shane M Liila-Fogarty, Grace E Boyum, Claire L Schwabe, Gaelen T Hess
Transcription factors (TFs) are a promising therapeutic target for a multitude of diseases. TFs perform their cellular roles by participating in multiple specific protein-protein interactions. For example, homo- or heterodimerization of some TFs controls DNA binding, while interactions between TFs and components of basal transcriptional machinery or chromatin modifiers can also be critical. While, in theory, small molecules could be used to disrupt specific protein-protein interfaces required for TF function, in practice, it is difficult to identify small molecules with the necessary specificity and efficacy, likely due to the extensive protein-protein interfaces that often underlie TF function. However, in contrast to small molecules, peptides have the potential to provide both the specificity and efficacy required to disrupt such interfaces. Here, we identified ∼15 peptides that inhibit the proliferation of leukemia cells using a high-throughput pooled screen of a library of 80-mer protein regions (peptides) derived from human nuclear-localized proteins. The antiproliferative peptides were enriched for regions known to be involved in specific TF dimerization, including the basic leucine zipper (bZIP) domain family. One of these bZIP domains, JDP2;bZIP_1, from the TF JDP2, was the top antiproliferative peptide, reducing the proliferation of K562 cells by 2-fold. JDP2;bZIP_1 inhibited AP-1 transcriptional activity and phenocopied JDP2 overexpression, suggesting that the peptide affected proliferation through a native JDP2 mechanism. Unexpectedly, given the strong conservation of the bZIP domain, residues outside of the annotated dimerization domain were critical for the peptide's antiproliferative potency. The peptide-mediated antiproliferative effect initiated erythrocyte differentiation in K562 cells and increased G0/G1 cells across multiple cell line models. We also found that many of the antiproliferative peptides identified in this study, including JDP2;bZIP_1, did not require a nuclear localization signal to function, a potential benefit for delivering these peptides in therapeutic applications.
{"title":"A High-Throughput Screen for Antiproliferative Peptides in Mammalian Cells Identifies Key Transcription Factor Families.","authors":"Shane M Liila-Fogarty, Grace E Boyum, Claire L Schwabe, Gaelen T Hess","doi":"10.1021/acssynbio.4c00337","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00337","url":null,"abstract":"<p><p>Transcription factors (TFs) are a promising therapeutic target for a multitude of diseases. TFs perform their cellular roles by participating in multiple specific protein-protein interactions. For example, homo- or heterodimerization of some TFs controls DNA binding, while interactions between TFs and components of basal transcriptional machinery or chromatin modifiers can also be critical. While, in theory, small molecules could be used to disrupt specific protein-protein interfaces required for TF function, in practice, it is difficult to identify small molecules with the necessary specificity and efficacy, likely due to the extensive protein-protein interfaces that often underlie TF function. However, in contrast to small molecules, peptides have the potential to provide both the specificity and efficacy required to disrupt such interfaces. Here, we identified ∼15 peptides that inhibit the proliferation of leukemia cells using a high-throughput pooled screen of a library of 80-mer protein regions (peptides) derived from human nuclear-localized proteins. The antiproliferative peptides were enriched for regions known to be involved in specific TF dimerization, including the basic leucine zipper (bZIP) domain family. One of these bZIP domains, JDP2;bZIP_1, from the TF JDP2, was the top antiproliferative peptide, reducing the proliferation of K562 cells by 2-fold. JDP2;bZIP_1 inhibited AP-1 transcriptional activity and phenocopied JDP2 overexpression, suggesting that the peptide affected proliferation through a native JDP2 mechanism. Unexpectedly, given the strong conservation of the bZIP domain, residues outside of the annotated dimerization domain were critical for the peptide's antiproliferative potency. The peptide-mediated antiproliferative effect initiated erythrocyte differentiation in K562 cells and increased G0/G1 cells across multiple cell line models. We also found that many of the antiproliferative peptides identified in this study, including JDP2;bZIP_1, did not require a nuclear localization signal to function, a potential benefit for delivering these peptides in therapeutic applications.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142453361","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-18Epub Date: 2024-10-02DOI: 10.1021/acssynbio.4c00360
Han Xiao, Hengrui Hu, Yijia Guo, Jiang Li, Wen-Bo Zeng, Min-Hua Luo, Manli Wang, Zhihong Hu
Synthesizing viral genomes plays an important role in fundamental virology research and in the development of vaccines and antiviral drugs. Herpes simplex virus type 1 (HSV-1) is a large DNA virus widely used in oncolytic virotherapy. Although de novo synthesis of the HSV-1 genome has been previously reported, the synthetic procedure is still far from efficient, and the synthesized genome contains a vector sequence that may affect its replication and application. In the present study, we developed an efficient vector-free strategy for synthesis and rescue of synthetic HSV-1. In contrast to the conventional method of transfecting mammalian cells with a completely synthesized genome containing a vector, overlapping HSV-1 fragments synthesized by transformation-associated recombination (TAR) in yeast were linearized and cotransfected into mammalian cells to rescue the synthetic virus. Using this strategy, a synthetic virus, F-Syn, comprising the complete genome of the HSV-1 F strain, was generated. The growth curve and electron microscopy of F-Syn confirmed that its replication dynamics and morphogenesis are similar to those of the parental virus. In addition, by combining TAR with in vitro CRISPR/Cas9 editing, an oncolytic virus, F-Syn-O, with deleted viral genes ICP6, ICP34.5, and ICP47 was generated. The antitumor effect of F-Syn-O was tested in vitro. F-Syn-O established a successful infection and induced dose-dependent cytotoxic effects in various human tumor cell lines. These strategies will facilitate convenient and systemic manipulation of HSV-1 genomes and could be further applied to the design and construction of oncolytic herpesviruses.
合成病毒基因组在病毒学基础研究以及疫苗和抗病毒药物的开发中发挥着重要作用。1 型单纯疱疹病毒(HSV-1)是一种大型 DNA 病毒,被广泛用于溶瘤病毒疗法。虽然此前已有从头合成 HSV-1 基因组的报道,但合成过程仍远不够高效,而且合成的基因组含有载体序列,可能会影响其复制和应用。在本研究中,我们开发了一种高效的无载体合成和挽救合成 HSV-1 的策略。与用含有载体的完全合成基因组转染哺乳动物细胞的传统方法不同,我们将在酵母中通过转化相关重组(TAR)合成的重叠 HSV-1 片段线性化并共转染到哺乳动物细胞中,以挽救合成病毒。利用这种策略,产生了一种合成病毒 F-Syn,它包含 HSV-1 F 株的完整基因组。F-Syn 的生长曲线和电子显微镜证实,其复制动态和形态发生与亲本病毒相似。此外,通过将TAR与体外CRISPR/Cas9编辑相结合,产生了一种删除了病毒基因ICP6、ICP34.5和ICP47的溶瘤病毒F-Syn-O。体外测试了 F-Syn-O 的抗肿瘤效果。F-Syn-O 成功感染了多种人类肿瘤细胞系,并诱导了剂量依赖性细胞毒性效应。这些策略将有助于对 HSV-1 基因组进行便捷、系统的操作,并可进一步应用于设计和构建溶瘤疱疹病毒。
{"title":"Efficient Strategy for Synthesizing Vector-Free and Oncolytic Herpes Simplex Type 1 Viruses.","authors":"Han Xiao, Hengrui Hu, Yijia Guo, Jiang Li, Wen-Bo Zeng, Min-Hua Luo, Manli Wang, Zhihong Hu","doi":"10.1021/acssynbio.4c00360","DOIUrl":"10.1021/acssynbio.4c00360","url":null,"abstract":"<p><p>Synthesizing viral genomes plays an important role in fundamental virology research and in the development of vaccines and antiviral drugs. Herpes simplex virus type 1 (HSV-1) is a large DNA virus widely used in oncolytic virotherapy. Although <i>de novo</i> synthesis of the HSV-1 genome has been previously reported, the synthetic procedure is still far from efficient, and the synthesized genome contains a vector sequence that may affect its replication and application. In the present study, we developed an efficient vector-free strategy for synthesis and rescue of synthetic HSV-1. In contrast to the conventional method of transfecting mammalian cells with a completely synthesized genome containing a vector, overlapping HSV-1 fragments synthesized by transformation-associated recombination (TAR) in yeast were linearized and cotransfected into mammalian cells to rescue the synthetic virus. Using this strategy, a synthetic virus, F-Syn, comprising the complete genome of the HSV-1 F strain, was generated. The growth curve and electron microscopy of F-Syn confirmed that its replication dynamics and morphogenesis are similar to those of the parental virus. In addition, by combining TAR with in vitro CRISPR/Cas9 editing, an oncolytic virus, F-Syn-O, with deleted viral genes <i>ICP6</i>, <i>ICP34.5</i>, and <i>ICP47</i> was generated. The antitumor effect of F-Syn-O was tested in vitro. F-Syn-O established a successful infection and induced dose-dependent cytotoxic effects in various human tumor cell lines. These strategies will facilitate convenient and systemic manipulation of HSV-1 genomes and could be further applied to the design and construction of oncolytic herpesviruses.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142363418","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-18Epub Date: 2024-09-22DOI: 10.1021/acssynbio.4c00219
Wouter Demeester, Brecht De Paepe, Marjan De Mey
LysR-type transcriptional regulators (LTTRs) are emerging as a promising group of macromolecules for the field of biosensors. As the largest family of bacterial transcription factors, the LTTRs represent a vast and mostly untapped repertoire of sensor proteins. To fully harness these regulators for transcription factor-based biosensor development, it is crucial to understand their underlying mechanisms and functionalities. In the first part, this Review discusses the established model and features of LTTRs. As dual-function regulators, these inducible transcription factors exude precise control over their regulatory targets. In the second part of this Review, an overview is given of the exceptions to the "classic" LTTR model. While a general regulatory mechanism has helped elucidate the intricate regulation performed by LTTRs, it is essential to recognize the variations within the family. By combining this knowledge, characterization of new regulators can be done more efficiently and accurately, accelerating the expansion of transcriptional sensors for biosensor development. Unlocking the pool of LTTRs would significantly expand the currently limited range of detectable molecules and regulatory functions available for the implementation of novel synthetic genetic circuitry.
{"title":"Fundamentals and Exceptions of the LysR-type Transcriptional Regulators.","authors":"Wouter Demeester, Brecht De Paepe, Marjan De Mey","doi":"10.1021/acssynbio.4c00219","DOIUrl":"10.1021/acssynbio.4c00219","url":null,"abstract":"<p><p>LysR-type transcriptional regulators (LTTRs) are emerging as a promising group of macromolecules for the field of biosensors. As the largest family of bacterial transcription factors, the LTTRs represent a vast and mostly untapped repertoire of sensor proteins. To fully harness these regulators for transcription factor-based biosensor development, it is crucial to understand their underlying mechanisms and functionalities. In the first part, this Review discusses the established model and features of LTTRs. As dual-function regulators, these inducible transcription factors exude precise control over their regulatory targets. In the second part of this Review, an overview is given of the exceptions to the \"classic\" LTTR model. While a general regulatory mechanism has helped elucidate the intricate regulation performed by LTTRs, it is essential to recognize the variations within the family. By combining this knowledge, characterization of new regulators can be done more efficiently and accurately, accelerating the expansion of transcriptional sensors for biosensor development. Unlocking the pool of LTTRs would significantly expand the currently limited range of detectable molecules and regulatory functions available for the implementation of novel synthetic genetic circuitry.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142277245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-18Epub Date: 2024-09-23DOI: 10.1021/acssynbio.4c00260
Tyson Bookout, Steve Shideler, Evan Cooper, Kira Goff, John V Headley, Lisa M Gieg, Shawn Lewenza
After extraction of bitumen from oil sands deposits, the oil sand process-affected water (OSPW) is stored in tailings ponds. Naphthenic acids (NA) in tailings ponds have been identified as the primary contributor to toxicity to aquatic life. As an alternative to other analytical methods, here we identify bacterial genes induced after growth in naphthenic acids and use synthetic biology approaches to construct a panel of candidate biosensors for NA detection in water. The main promoters of interest were the atuAR promoters from a naphthenic acid degradation operon and upstream TetR regulator, the marR operon which includes a MarR regulator and downstream naphthenic acid resistance genes, and a hypothetical gene with a possible role in fatty acid biology. Promoters were printed and cloned as transcriptional lux reporter plasmids that were introduced into a tailings pond-derived Pseudomonas species. All candidate biosensor strains were tested for transcriptional responses to naphthenic acid mixtures and individual compounds. The three priority promoters respond in a dose-dependent manner to simple, acyclic, and complex NA mixtures, and each promoter has unique NA specificities. The limits of NA detection from the various NA mixtures ranged between 1.5 and 15 mg/L. The atuA and marR promoters also detected NA in small volumes of OSPW samples and were induced by extracts of the panel of OSPW samples. While biosensors have been constructed for other hydrocarbons, here we describe a biosensor approach that could be employed in environmental monitoring of naphthenic acids in oil sands mining wastewater.
从油砂矿床中提取沥青后,受油砂加工影响的水(OSPW)被储存在尾矿池中。尾矿池中的环烷酸(NA)已被确定为造成水生生物毒性的主要因素。作为其他分析方法的替代方法,我们在此确定了在环烷酸中生长后诱导的细菌基因,并使用合成生物学方法构建了一组用于检测水中 NA 的候选生物传感器。我们感兴趣的主要启动子是来自环烷酸降解操作子和上游 TetR 调节器的 atuAR 启动子、包括 MarR 调节器和下游环烷酸抗性基因的 marR 操作子,以及一个可能在脂肪酸生物学中发挥作用的假定基因。启动子被打印出来并克隆为转录勒克斯报告质粒,引入尾矿库衍生的假单胞菌物种中。测试了所有候选生物传感器菌株对环烷酸混合物和单个化合物的转录反应。三个优先启动子以剂量依赖的方式对简单、非环状和复杂的环烷酸混合物做出反应,每个启动子都有独特的环烷酸特异性。各种 NA 混合物的 NA 检测限在 1.5 至 15 mg/L 之间。atuA和marR启动子也能在小体积的OSPW样本中检测到NA,并且能被OSPW样本的提取物所诱导。虽然生物传感器是针对其他碳氢化合物构建的,但我们在此介绍一种可用于油砂开采废水中环烷酸环境监测的生物传感器方法。
{"title":"Construction of Whole Cell Bacterial Biosensors as an Alternative Environmental Monitoring Technology to Detect Naphthenic Acids in Oil Sands Process-Affected Water.","authors":"Tyson Bookout, Steve Shideler, Evan Cooper, Kira Goff, John V Headley, Lisa M Gieg, Shawn Lewenza","doi":"10.1021/acssynbio.4c00260","DOIUrl":"10.1021/acssynbio.4c00260","url":null,"abstract":"<p><p>After extraction of bitumen from oil sands deposits, the oil sand process-affected water (OSPW) is stored in tailings ponds. Naphthenic acids (NA) in tailings ponds have been identified as the primary contributor to toxicity to aquatic life. As an alternative to other analytical methods, here we identify bacterial genes induced after growth in naphthenic acids and use synthetic biology approaches to construct a panel of candidate biosensors for NA detection in water. The main promoters of interest were the <i>atuAR</i> promoters from a naphthenic acid degradation operon and upstream TetR regulator, the <i>marR</i> operon which includes a MarR regulator and downstream naphthenic acid resistance genes, and a hypothetical gene with a possible role in fatty acid biology. Promoters were printed and cloned as transcriptional <i>lux</i> reporter plasmids that were introduced into a tailings pond-derived <i>Pseudomonas</i> species. All candidate biosensor strains were tested for transcriptional responses to naphthenic acid mixtures and individual compounds. The three priority promoters respond in a dose-dependent manner to simple, acyclic, and complex NA mixtures, and each promoter has unique NA specificities. The limits of NA detection from the various NA mixtures ranged between 1.5 and 15 mg/L. The <i>atuA</i> and <i>marR</i> promoters also detected NA in small volumes of OSPW samples and were induced by extracts of the panel of OSPW samples. While biosensors have been constructed for other hydrocarbons, here we describe a biosensor approach that could be employed in environmental monitoring of naphthenic acids in oil sands mining wastewater.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142306532","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Drosophila melanogaster (fruit fly) is an animal model chassis in biological and genetic research owing to its short life cycle, ease of cultivation, and acceptability to genetic modification. While the D. melanogaster chassis offers valuable insights into drug efficacy, toxicity, and mechanisms, several obvious challenges such as dosage control and drug resistance still limit its utility in pharmacological studies. Our research combines optogenetic control with engineered gut bacteria to facilitate the precise delivery of therapeutic substances in D. melanogaster for biomedical research. We have shown that the engineered bacteria can be orally administered to D. melanogaster to get a stable density of approximately 28,000 CFUs/per fly, leading to no detectable negative effects on the growth of D. melanogaster. In a model of D. melanogaster exposure to heavy metal, these orally administered bacteria uniformly express target genes under green light control to produce MtnB protein for binding and detoxifying lead, which significantly reduces the level of oxidative stress in the intestinal tract of Pb-treated flies. This pioneering study lays the groundwork for using optogenetic-controlled bacteria in the model chassis D. melanogaster to advance biomedical applications.
{"title":"Implementing Optogenetic-Controlled Bacterial Systems in <i>Drosophila melanogaster</i> for Alleviation of Heavy Metal Poisoning.","authors":"Junyi Wang, Ying Li, Dawei Sun, Jingyi Li, Lianyue Li, Xinyu Zhang, Xinyu Liu, Zhijie Feng, Huimin Xue, Yuhui Cui, Yiwen Wang, Duo Liu, Hanjie Wang","doi":"10.1021/acssynbio.4c00409","DOIUrl":"10.1021/acssynbio.4c00409","url":null,"abstract":"<p><p><i>Drosophila melanogaster</i> (fruit fly) is an animal model chassis in biological and genetic research owing to its short life cycle, ease of cultivation, and acceptability to genetic modification. While the <i>D. melanogaster</i> chassis offers valuable insights into drug efficacy, toxicity, and mechanisms, several obvious challenges such as dosage control and drug resistance still limit its utility in pharmacological studies. Our research combines optogenetic control with engineered gut bacteria to facilitate the precise delivery of therapeutic substances in <i>D. melanogaster</i> for biomedical research. We have shown that the engineered bacteria can be orally administered to <i>D. melanogaster</i> to get a stable density of approximately 28,000 CFUs/per fly, leading to no detectable negative effects on the growth of <i>D. melanogaster</i>. In a model of <i>D. melanogaster</i> exposure to heavy metal, these orally administered bacteria uniformly express target genes under green light control to produce MtnB protein for binding and detoxifying lead, which significantly reduces the level of oxidative stress in the intestinal tract of Pb-treated flies. This pioneering study lays the groundwork for using optogenetic-controlled bacteria in the model chassis <i>D. melanogaster</i> to advance biomedical applications.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142306536","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-18Epub Date: 2024-10-08DOI: 10.1021/acssynbio.4c00271
Yiming Wan, Christopher Helenek, Damiano Coraci, Gábor Balázsi
The invention of RNA-guided DNA cutting systems has revolutionized biotechnology. More recently, RNA-guided RNA cutting by Cas13d entered the scene as a highly promising alternative to RNA interference to engineer cellular transcriptomes for biotechnological and therapeutic purposes. Unfortunately, "collateral damage" by indiscriminate off-target cutting tampered enthusiasm for these systems. Yet, how collateral activity, or even RNA target reduction depends on Cas13d and guide RNA abundance has remained unclear due to the lack of expression-tuning studies to address this question. Here we use precise expression-tuning gene circuits to show that both nonspecific and specific, on-target RNA reduction depend on Cas13d and guide RNA levels, and that nonspecific RNA cutting from trans cleavage might contribute to on-target RNA reduction. Using RNA-level control techniques, we develop new Multi-Level Optimized Negative-Autoregulated Cas13d and crRNA Hybrid (MONARCH) gene circuits that achieve a high dynamic range with low basal on-target RNA reduction while minimizing collateral activity in human kidney cells and green monkey cells most frequently used in human virology. MONARCH should bring RNA-guided RNA cutting systems to the forefront, as easily applicable, programmable tools for transcriptome engineering in biotechnological and medical applications.
{"title":"Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting.","authors":"Yiming Wan, Christopher Helenek, Damiano Coraci, Gábor Balázsi","doi":"10.1021/acssynbio.4c00271","DOIUrl":"10.1021/acssynbio.4c00271","url":null,"abstract":"<p><p>The invention of RNA-guided DNA cutting systems has revolutionized biotechnology. More recently, RNA-guided RNA cutting by Cas13d entered the scene as a highly promising alternative to RNA interference to engineer cellular transcriptomes for biotechnological and therapeutic purposes. Unfortunately, \"collateral damage\" by indiscriminate off-target cutting tampered enthusiasm for these systems. Yet, how collateral activity, or even RNA target reduction depends on Cas13d and guide RNA abundance has remained unclear due to the lack of expression-tuning studies to address this question. Here we use precise expression-tuning gene circuits to show that both nonspecific and specific, on-target RNA reduction depend on Cas13d and guide RNA levels, and that nonspecific RNA cutting from <i>trans</i> cleavage might contribute to on-target RNA reduction. Using RNA-level control techniques, we develop new <i>Multi-Level Optimized Negative-Autoregulated Cas13d and crRNA Hybrid</i> (MONARCH) gene circuits that achieve a high dynamic range with low basal on-target RNA reduction while minimizing collateral activity in human kidney cells and green monkey cells most frequently used in human virology. MONARCH should bring RNA-guided RNA cutting systems to the forefront, as easily applicable, programmable tools for transcriptome engineering in biotechnological and medical applications.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142386364","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Stenotrophomonas maltophilia (S. maltophilia, SMA) is a common opportunistic pathogen that poses a serious threat to the food industry and human health. Traditional detection methods for SMA are time-consuming, have low detection rates, require complex and expensive equipment and professional technical personnel for operation, and are unsuitable for on-site detection. Therefore, establishing an efficient on-site detection method has great significance in formulating appropriate treatment strategies and ensuring food safety. In the present study, a rapid one-pot detection method was established for SMA using a combination of Recombinase Polymerase Amplification (RPA) and CRISPR/Cas12a, referred to as ORCas12a-SMA (one-pot RPA-CRISPR/Cas12a platform). In the ORCas12a-SMA detection method, all components were added into a single tube simultaneously to achieve one-pot detection and address the problems of nucleic acid cross-contamination and reduced sensitivity caused by frequent cap opening during stepwise detection. The ORCas12a-SMA method could detect at least 3 × 10° copies·μL-1 of SMA genomic DNA within 30 min at 37 °C. Additionally, this method exhibited sensitivity compared to the typical two-step RPA-CRISPR/Cas12a method. Overall, the ORCas12a-SMA detection offered the advantages of rapidity, simplicity, high sensitivity and specificity, and decreased need for complex large-scale instrumentation. This assay is the first application of the one-pot platform based on the combination of RPA and CRISPR/Cas12a in SMA detection and is highly suitable for point-of-care testing. It helps reduce losses in the food industry and provides assistance in formulating timely and appropriate antimicrobial treatment plans.
{"title":"One-Pot Assay for Rapid Detection of <i>Stenotrophomonas maltophilia</i> by RPA-CRISPR/Cas12a.","authors":"Jiangli Zhang, Ling Qin, Yingying Chang, Yulong He, Weichao Zhao, Yongyou Zhao, Yanan Ding, Jin Gao, Xiting Zhao","doi":"10.1021/acssynbio.4c00481","DOIUrl":"10.1021/acssynbio.4c00481","url":null,"abstract":"<p><p><i>Stenotrophomonas maltophilia</i> (<i>S. maltophilia</i>, SMA) is a common opportunistic pathogen that poses a serious threat to the food industry and human health. Traditional detection methods for SMA are time-consuming, have low detection rates, require complex and expensive equipment and professional technical personnel for operation, and are unsuitable for on-site detection. Therefore, establishing an efficient on-site detection method has great significance in formulating appropriate treatment strategies and ensuring food safety. In the present study, a rapid one-pot detection method was established for SMA using a combination of Recombinase Polymerase Amplification (RPA) and CRISPR/Cas12a, referred to as ORCas12a-SMA (one-pot RPA-CRISPR/Cas12a platform). In the ORCas12a-SMA detection method, all components were added into a single tube simultaneously to achieve one-pot detection and address the problems of nucleic acid cross-contamination and reduced sensitivity caused by frequent cap opening during stepwise detection. The ORCas12a-SMA method could detect at least 3 × 10° copies·μL<sup>-1</sup> of SMA genomic DNA within 30 min at 37 °C. Additionally, this method exhibited sensitivity compared to the typical two-step RPA-CRISPR/Cas12a method. Overall, the ORCas12a-SMA detection offered the advantages of rapidity, simplicity, high sensitivity and specificity, and decreased need for complex large-scale instrumentation. This assay is the first application of the one-pot platform based on the combination of RPA and CRISPR/Cas12a in SMA detection and is highly suitable for point-of-care testing. It helps reduce losses in the food industry and provides assistance in formulating timely and appropriate antimicrobial treatment plans.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142363419","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-18DOI: 10.1021/acssynbio.4c00347
Yu Tian, Liwen Yang, Shaozhen Ding, Dachuan Zhang, Le Yuan, Zhiguo Liu, Qian-Nan Hu
Synthetic biology is rapidly evolving into a data-intensive science that increasingly relies on massive data sets; one of its applications is the evaluation of the economic viability of fermentation processes. However, the key economic indicators, namely titer, rate, and yield (TRY), which respectively reflect the downstream processing, reactor size, and raw material costs, are not well captured in bioinformatics databases. In this paper, we present BioTRY, an intuitive and user-friendly tool that contains >5,000 biochemicals and >3,800 strains, along with over 52,000 corresponding TRY entries with original references. It is freely available at http://www.synbiohealth.cn/biotry. To our knowledge, BioTRY is the first available database on biosynthesis TRY data from original research. We anticipate that BioTRY will become a useful tool that aids researchers and decision-makers in understanding the current development state of biosynthesis and allows them to foresee potential prospects and applications for biosynthesis.
{"title":"BioTRY: A Comprehensive Knowledge Base for Titer, Rate, and Yield of Biosynthesis.","authors":"Yu Tian, Liwen Yang, Shaozhen Ding, Dachuan Zhang, Le Yuan, Zhiguo Liu, Qian-Nan Hu","doi":"10.1021/acssynbio.4c00347","DOIUrl":"https://doi.org/10.1021/acssynbio.4c00347","url":null,"abstract":"<p><p>Synthetic biology is rapidly evolving into a data-intensive science that increasingly relies on massive data sets; one of its applications is the evaluation of the economic viability of fermentation processes. However, the key economic indicators, namely titer, rate, and yield (TRY), which respectively reflect the downstream processing, reactor size, and raw material costs, are not well captured in bioinformatics databases. In this paper, we present BioTRY, an intuitive and user-friendly tool that contains >5,000 biochemicals and >3,800 strains, along with over 52,000 corresponding TRY entries with original references. It is freely available at http://www.synbiohealth.cn/biotry. To our knowledge, BioTRY is the first available database on biosynthesis TRY data from original research. We anticipate that BioTRY will become a useful tool that aids researchers and decision-makers in understanding the current development state of biosynthesis and allows them to foresee potential prospects and applications for biosynthesis.</p>","PeriodicalId":26,"journal":{"name":"ACS Synthetic Biology","volume":null,"pages":null},"PeriodicalIF":3.7,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142453363","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}