Pub Date : 2024-08-06DOI: 10.1101/2024.08.06.606454
Leo Scheller, Greta Maria Paola Giordano Attianese, Rocío Castellanos-Rueda, Raphaël B Di Roberto, Markus Barden, Melanie Triboulet, Sailan Shui, Elisabetta Cribioli, Anthony Marchand, Sandrine Georgeon, Hinrich Abken, Sai Reddy, Bruno E. Correia, Melita Irving
CAR T-cell therapy is constrained by on-target, off-tumor toxicities as well as cellular exhaustion due to chronic antigen exposure. CARs comprising small-molecule controlled switches can enhance both safety and therapeutic efficacy but are limited by the scarcity of non-immunogenic protein elements responsive to non-immunosuppressive, clinically approved drugs with favorable pharmacodynamics. Here, we combine rational design and library-based optimization of a protein-protein interaction (PPI) of human origin to develop venetoclax-controlled Drug-Regulated Off-switch PPI (DROP)-CARs. DROP-CARs enable dose-dependent release of the tumor-targeting scFv and consequent T-cell dissociation from the target tumor cell. Additionally, we present proof-of-concept for a dual DROP-CAR controlled by different small molecules, as well as for logic-gated synthetic receptors enabling STAT3 signaling. We demonstrate in vitro and in vivo function of DROP-CAR T cells and conclude that the approach holds promise for clinical application.
CAR T 细胞疗法受到靶上、瘤外毒性以及长期抗原暴露导致的细胞衰竭的限制。由小分子可控开关组成的 CAR 可以提高安全性和疗效，但由于缺乏对非免疫抑制性、临床批准的药物有良好药效学反应的非免疫原性蛋白元素，因此受到限制。在这里，我们将人类蛋白-蛋白相互作用（PPI）的合理设计和基于文库的优化结合起来，开发出了由 Venetoclax 控制的药物调节关开关 PPI（DROP）-CARs。DROP-CARs 能使肿瘤靶向 scFv 按剂量释放，从而使 T 细胞与靶肿瘤细胞分离。此外，我们还展示了由不同小分子控制的双 DROP-CAR 概念验证，以及可实现 STAT3 信号传导的逻辑门控合成受体。我们展示了 DROP-CAR T 细胞的体外和体内功能，并得出结论：这种方法有望应用于临床。
{"title":"Drug-controlled CAR-T cells through the regulation of cell-cell interactions","authors":"Leo Scheller, Greta Maria Paola Giordano Attianese, Rocío Castellanos-Rueda, Raphaël B Di Roberto, Markus Barden, Melanie Triboulet, Sailan Shui, Elisabetta Cribioli, Anthony Marchand, Sandrine Georgeon, Hinrich Abken, Sai Reddy, Bruno E. Correia, Melita Irving","doi":"10.1101/2024.08.06.606454","DOIUrl":"https://doi.org/10.1101/2024.08.06.606454","url":null,"abstract":"CAR T-cell therapy is constrained by on-target, off-tumor toxicities as well as cellular exhaustion due to chronic antigen exposure. CARs comprising small-molecule controlled switches can enhance both safety and therapeutic efficacy but are limited by the scarcity of non-immunogenic protein elements responsive to non-immunosuppressive, clinically approved drugs with favorable pharmacodynamics. Here, we combine rational design and library-based optimization of a protein-protein interaction (PPI) of human origin to develop venetoclax-controlled Drug-Regulated Off-switch PPI (DROP)-CARs. DROP-CARs enable dose-dependent release of the tumor-targeting scFv and consequent T-cell dissociation from the target tumor cell. Additionally, we present proof-of-concept for a dual DROP-CAR controlled by different small molecules, as well as for logic-gated synthetic receptors enabling STAT3 signaling. We demonstrate in vitro and in vivo function of DROP-CAR T cells and conclude that the approach holds promise for clinical application.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"33 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141948031","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-05DOI: 10.1101/2024.08.05.606709
Ana Pascual-Garrigos, Beatriz Lozano-Torres, Akashaditya Das, Jennifer C Molloy
There is a critical need to implement a sensitive and specific point-of-care (POC) biosensor that addresses the instrument limitations and manufacturing challenges faced in resource-constrained contexts. In this paper we focus on enteric fever which is a highly contagious and prevalent infection in low- and middle-income countries. Although easily treatable, its ambiguous symptoms paired with a lack of fast, accurate and affordable diagnostics lead to incorrect treatments which exacerbate the disease burden, including increasing antibiotic resistance. In this study, we develop a readout module for CRISPR-Cas12a that produces a colorimetric output that is visible to the naked eye and can act as a cascade signal amplifier in any CRISPR assay based on trans-cleavage. We achieve this by immobilizing an oligo covalently linked to a β-galactosidase (LacZ) enzyme, which is cleaved in the presence of DNA target-activated CRISPR-Cas12a. Upon cleavage, the colorimetric enzyme is released, and the supernatant transferred to an environment containing X-Gal producing an intense blue color. This method is capable of detecting amplified bacterial genomic DNA and has a lower limit of detection (LoD) to standard fluorescent assays while removing the requirement for costly equipment. Furthermore, it remained active after lyophilization, allowing for the possibility of shipment without cold chain, significantly reducing deployment costs.
{"title":"Colorimetric CRISPR Biosensor: A Case Study with Salmonella Typhi","authors":"Ana Pascual-Garrigos, Beatriz Lozano-Torres, Akashaditya Das, Jennifer C Molloy","doi":"10.1101/2024.08.05.606709","DOIUrl":"https://doi.org/10.1101/2024.08.05.606709","url":null,"abstract":"There is a critical need to implement a sensitive and specific point-of-care (POC) biosensor that addresses the instrument limitations and manufacturing challenges faced in resource-constrained contexts. In this paper we focus on enteric fever which is a highly contagious and prevalent infection in low- and middle-income countries. Although easily treatable, its ambiguous symptoms paired with a lack of fast, accurate and affordable diagnostics lead to incorrect treatments which exacerbate the disease burden, including increasing antibiotic resistance. In this study, we develop a readout module for CRISPR-Cas12a that produces a colorimetric output that is visible to the naked eye and can act as a cascade signal amplifier in any CRISPR assay based on trans-cleavage. We achieve this by immobilizing an oligo covalently linked to a β-galactosidase (LacZ) enzyme, which is cleaved in the presence of DNA target-activated CRISPR-Cas12a. Upon cleavage, the colorimetric enzyme is released, and the supernatant transferred to an environment containing X-Gal producing an intense blue color. This method is capable of detecting amplified bacterial genomic DNA and has a lower limit of detection (LoD) to standard fluorescent assays while removing the requirement for costly equipment. Furthermore, it remained active after lyophilization, allowing for the possibility of shipment without cold chain, significantly reducing deployment costs.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"69 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141948030","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
DNA information storage provides an excellent solution for metadata storage due to its high density, programmability, and long-term stability. However, current research in DNA storage primarily focuses on the processes of storing and reading data, lacking comprehensive solutions for the secure metadata wiping. Herein, we present a method of random sanitization in DNA information storage using CRISPR-Cas12a (RSDISC) based on precise control of the thermodynamic energy of primer-template hybridization. We utilize the collateral cleavage (trans-activity) of single-stranded DNA (ssDNA) by CRISPR-Cas12a to achieve selective sanitization of files in metadata. This method enables ssDNA degradation with different GC content, lengths, and secondary structures to achieve a sanitization efficiency up to 99.9% for 28,258 oligonucleotides in DNA storage within one round. We demonstrate that the number of erasable files could reach 1011.7 based on a model of primer-template hybridization efficiency. Overall, RSDISC provides a random sanitization approach to set the foundation of information encryption, file classification, memory deallocation and accurate reading in DNA data storage.
DNA 信息存储因其高密度、可编程性和长期稳定性，为元数据存储提供了极佳的解决方案。然而，目前DNA存储的研究主要集中在存储和读取数据的过程，缺乏安全抹除元数据的全面解决方案。在此，我们提出了一种利用CRISPR-Cas12a（RSDISC）在DNA信息存储中进行随机清除的方法，该方法基于对引物-模板杂交热力学能量的精确控制。我们利用 CRISPR-Cas12a 对单链 DNA（ssDNA）的附带裂解（反式活性）来实现元数据中文件的选择性消毒。这种方法可以降解具有不同 GC 含量、长度和二级结构的 ssDNA，在一轮内对 DNA 存储中的 28,258 个寡核苷酸实现高达 99.9% 的消毒效率。根据引物-模板杂交效率模型，我们证明可擦除文件的数量可达 1011.7 个。总之，RSDISC 提供了一种随机清除方法，为 DNA 数据存储中的信息加密、文件分类、内存删除和精确读取奠定了基础。
{"title":"Random Sanitization in DNA information storage using CRISPR-Cas12a","authors":"Hongyu Shen, Zhi Weng, Haipei Zhao, Haitao Song, Fei Wang, Chunhai Fan, Ping Song","doi":"10.1101/2024.08.04.606549","DOIUrl":"https://doi.org/10.1101/2024.08.04.606549","url":null,"abstract":"DNA information storage provides an excellent solution for metadata storage due to its high density, programmability, and long-term stability. However, current research in DNA storage primarily focuses on the processes of storing and reading data, lacking comprehensive solutions for the secure metadata wiping. Herein, we present a method of random sanitization in DNA information storage using CRISPR-Cas12a (RSDISC) based on precise control of the thermodynamic energy of primer-template hybridization. We utilize the collateral cleavage (trans-activity) of single-stranded DNA (ssDNA) by CRISPR-Cas12a to achieve selective sanitization of files in metadata. This method enables ssDNA degradation with different GC content, lengths, and secondary structures to achieve a sanitization efficiency up to 99.9% for 28,258 oligonucleotides in DNA storage within one round. We demonstrate that the number of erasable files could reach 1011.7 based on a model of primer-template hybridization efficiency. Overall, RSDISC provides a random sanitization approach to set the foundation of information encryption, file classification, memory deallocation and accurate reading in DNA data storage.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"77 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141969772","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-02DOI: 10.1101/2024.07.30.605672
Grant M Landwehr, Jonathan W Bogart, Carol Magalhaes, Eric Hammarlund, Ashty S Karim, Michael C Jewett
Enzyme engineering is limited by the challenge of rapidly generating and using large datasets of sequence-function relationships for predictive design. To address this challenge, we developed a machine learning (ML)-guided platform that integrates cell-free DNA assembly, cell-free gene expression, and functional assays to rapidly map fitness landscapes across protein sequence space and optimize enzymes for multiple, distinct chemical reactions. We applied this platform to engineer amide synthetases by evaluating substrate preference for 1,217 enzyme variants in 10,953 unique reactions. We used these data to build augmented ridge regression ML models for predicting amide synthetase variants capable of making 9 small molecule pharmaceuticals. Our ML-guided, cell-free framework promises to accelerate enzyme engineering by enabling iterative exploration of protein sequence space to build specialized biocatalysts in parallel.
酶工程受限于快速生成和使用大量序列-功能关系数据集进行预测性设计的挑战。为了应对这一挑战，我们开发了一个机器学习（ML）指导的平台，该平台整合了无细胞 DNA 组装、无细胞基因表达和功能检测，可快速绘制整个蛋白质序列空间的适应性景观，并针对多种不同的化学反应优化酶。我们将该平台应用于酰胺合成酶的工程化，评估了 10953 个独特反应中 1,217 个酶变体的底物偏好。我们利用这些数据建立了增强脊回归 ML 模型，用于预测能够制造 9 种小分子药物的酰胺合成酶变体。我们的以 ML 为指导的无细胞框架有望通过迭代探索蛋白质序列空间来并行构建专门的生物催化剂，从而加速酶工程。
{"title":"Accelerated enzyme engineering by machine-learning guided cell-free expression","authors":"Grant M Landwehr, Jonathan W Bogart, Carol Magalhaes, Eric Hammarlund, Ashty S Karim, Michael C Jewett","doi":"10.1101/2024.07.30.605672","DOIUrl":"https://doi.org/10.1101/2024.07.30.605672","url":null,"abstract":"Enzyme engineering is limited by the challenge of rapidly generating and using large datasets of sequence-function relationships for predictive design. To address this challenge, we developed a machine learning (ML)-guided platform that integrates cell-free DNA assembly, cell-free gene expression, and functional assays to rapidly map fitness landscapes across protein sequence space and optimize enzymes for multiple, distinct chemical reactions. We applied this platform to engineer amide synthetases by evaluating substrate preference for 1,217 enzyme variants in 10,953 unique reactions. We used these data to build augmented ridge regression ML models for predicting amide synthetase variants capable of making 9 small molecule pharmaceuticals. Our ML-guided, cell-free framework promises to accelerate enzyme engineering by enabling iterative exploration of protein sequence space to build specialized biocatalysts in parallel.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"32 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141883760","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-01DOI: 10.1101/2024.08.01.606163
Atsuko Uenoyama, Hana Kiyama, Mone Mimura, Makoto Miyata
JCVI-syn3B (syn3B), a minimal synthetic bacterium that only possesses essential genes, facilitates the examination of heterogeneous gene functions in minimal life. Conventionally, Escherichia coli is used to construct DNA fragments for gene transfer into the syn3B genome. However, the construction process is challenging and time-consuming due to various issues, including the inhibition of E. coli growth and unexpected recombination, especially with AT-rich DNA sequences such as those found in Mycoplasma genes. Therefore, in this study, we aimed to develop a new transformation method to overcome these issues. We assembled the vector and target DNA fragments using an in vitro homologous recombination system and subsequently transferred the products into the syn3B genome. We obtained approximately 5,000 recombinant colonies per milliliter of the original culture in eight days, which is four days shorter than the conventional period, without any recombination issues, even for AT-rich DNA
JCVI-syn3B（syn3B）是一种只拥有基本基因的最小合成细菌，有助于研究最小生命中的异质基因功能。传统方法是利用大肠杆菌构建 DNA 片段，将基因转移到 syn3B 基因组中。然而，由于大肠杆菌生长受到抑制和意外重组等各种问题，尤其是在富含 AT 的 DNA 序列（如支原体基因中发现的序列）中，构建过程具有挑战性且耗时较长。因此，在本研究中，我们旨在开发一种新的转化方法来克服这些问题。我们利用体外同源重组系统组装了载体和目标DNA片段，随后将产物转移到syn3B基因组中。我们在八天内获得了每毫升原始培养物约 5,000 个重组菌落，比传统方法缩短了四天，而且没有出现任何重组问题，即使是富含 AT 的 DNA
{"title":"Establishment of a rapid method to assemble and transfer DNA fragments into the JCVI-syn3B minimal synthetic bacterial genome","authors":"Atsuko Uenoyama, Hana Kiyama, Mone Mimura, Makoto Miyata","doi":"10.1101/2024.08.01.606163","DOIUrl":"https://doi.org/10.1101/2024.08.01.606163","url":null,"abstract":"JCVI-syn3B (syn3B), a minimal synthetic bacterium that only possesses essential genes, facilitates the examination of heterogeneous gene functions in minimal life. Conventionally, Escherichia coli is used to construct DNA fragments for gene transfer into the syn3B genome. However, the construction process is challenging and time-consuming due to various issues, including the inhibition of E. coli growth and unexpected recombination, especially with AT-rich DNA sequences such as those found in Mycoplasma genes. Therefore, in this study, we aimed to develop a new transformation method to overcome these issues. We assembled the vector and target DNA fragments using an in vitro homologous recombination system and subsequently transferred the products into the syn3B genome. We obtained approximately 5,000 recombinant colonies per milliliter of the original culture in eight days, which is four days shorter than the conventional period, without any recombination issues, even for AT-rich DNA","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"34 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141883761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-08-01DOI: 10.1101/2024.07.31.606098
Tong Chen, Yinuo Zhang, Pranam Chatterjee
The ability to precisely target specific motifs on disease-related proteins, whether conserved epitopes on viral proteins, intrinsically disordered regions within transcription factors, or breakpoint junctions in fusion oncoproteins, is essential for modulating their function while minimizing off-target effects. Current methods struggle to achieve this specificity without reliable structural information. In this work, we introduce a motif-specific PPItargeting algorithm, moPPIt, for de novo generation of motif-specific peptide binders from the target protein sequence alone. At the core of moPPIt is BindEvaluator, a transformer-based model that interpolates protein language model embeddings of two proteins via a series of multi-headed self-attention blocks, with a key focus on local motif features. Trained on over 510,000 annotated PPIs, BindEvaluator accurately predicts target binding sites given protein-protein sequence pairs with a test AUC > 0.94, improving to AUC > 0.96 when fine-tuned on peptide-protein pairs. By combining BindEvaluator with our PepMLM peptide generator and genetic algorithm-based optimization, moPPIt generates peptides that bind specifically to user-defined residues on target proteins. We demonstrate moPPIt's efficacy in computationally designing binders to specific motifs, first on targets with known binding peptides and then extending to structured and disordered targets with no known binders. In total, moPPIt serves as a powerful tool for developing highly specific peptide therapeutics without relying on target structure or structure-dependent latent spaces.
{"title":"moPPIt: De Novo Generation of Motif-Specific Binders with Protein Language Models","authors":"Tong Chen, Yinuo Zhang, Pranam Chatterjee","doi":"10.1101/2024.07.31.606098","DOIUrl":"https://doi.org/10.1101/2024.07.31.606098","url":null,"abstract":"The ability to precisely target specific motifs on disease-related proteins, whether conserved epitopes on viral proteins, intrinsically disordered regions within transcription factors, or breakpoint junctions in fusion oncoproteins, is essential for modulating their function while minimizing off-target effects. Current methods struggle to achieve this specificity without reliable structural information. In this work, we introduce a <strong>mo</strong>tif-specific <strong>PPI</strong> <strong>t</strong>argeting algorithm, <strong>moPPIt</strong>, for <em>de novo</em> generation of motif-specific peptide binders from the target protein sequence alone. At the core of moPPIt is BindEvaluator, a transformer-based model that interpolates protein language model embeddings of two proteins via a series of multi-headed self-attention blocks, with a key focus on local motif features. Trained on over 510,000 annotated PPIs, BindEvaluator accurately predicts target binding sites given protein-protein sequence pairs with a test AUC > 0.94, improving to AUC > 0.96 when fine-tuned on peptide-protein pairs. By combining BindEvaluator with our PepMLM peptide generator and genetic algorithm-based optimization, moPPIt generates peptides that bind specifically to user-defined residues on target proteins. We demonstrate moPPIt's efficacy in computationally designing binders to specific motifs, first on targets with known binding peptides and then extending to structured and disordered targets with no known binders. In total, moPPIt serves as a powerful tool for developing highly specific peptide therapeutics without relying on target structure or structure-dependent latent spaces.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141883762","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-31DOI: 10.1101/2024.07.31.606084
Cody Kamoku, Cheyanna Cooper, Ashley Straub, Nathan Miller, David R Nielsen
In most cyanobacteria, genetic engineering efforts currently rely upon chromosomal integration; a time-consuming process due to their polyploid nature. To enhance strain construction, here we develop and characterize two novel replicating plasmids for use in Synechococcus sp. PCC 7002. Following an initial screen of plasmids comprising seven different origins of replication, two were found capable of replication: one based on the WVO1 broad host range plasmid and the other a shuttle vector derived from pCB2.4 from Synechocystis sp. PCC 6803. These were then used to construct a set of new replicating plasmids, which were shown to be both co-transformable and stably maintained in PCC 7002 at copy numbers between 0.6-1.4 and 7-16, respectively. Lastly, we demonstrate the importance of using multimeric plasmids during natural transformation of PCC 7002, with higher order multimers providing a 30-fold increase in transformation efficiency relative to monomeric plasmids. Useful considerations and methods for enhancing multimer content in plasmid samples are also presented.
{"title":"Delivery of novel replicating vectors to Synechococcus sp. PCC 7002 via natural transformation of plasmid multimers","authors":"Cody Kamoku, Cheyanna Cooper, Ashley Straub, Nathan Miller, David R Nielsen","doi":"10.1101/2024.07.31.606084","DOIUrl":"https://doi.org/10.1101/2024.07.31.606084","url":null,"abstract":"In most cyanobacteria, genetic engineering efforts currently rely upon chromosomal integration; a time-consuming process due to their polyploid nature. To enhance strain construction, here we develop and characterize two novel replicating plasmids for use in <em>Synechococcus</em> sp. PCC 7002. Following an initial screen of plasmids comprising seven different origins of replication, two were found capable of replication: one based on the WVO1 broad host range plasmid and the other a shuttle vector derived from pCB2.4 from <em>Synechocystis</em> sp. PCC 6803. These were then used to construct a set of new replicating plasmids, which were shown to be both co-transformable and stably maintained in PCC 7002 at copy numbers between 0.6-1.4 and 7-16, respectively. Lastly, we demonstrate the importance of using multimeric plasmids during natural transformation of PCC 7002, with higher order multimers providing a 30-fold increase in transformation efficiency relative to monomeric plasmids. Useful considerations and methods for enhancing multimer content in plasmid samples are also presented.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"10 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141883764","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-31DOI: 10.1101/2024.07.31.606091
Lubica Supekova, Han Zhou, Izabella H. Barcellos, Catherine Nguyen, David A. Dik, Peter G. Schultz
Bacteria engulfment by a higher order host is believed to be the beginning of an evolutionary process that ultimately formed mitochondria. In an effort to experimentally elucidate the early effects of natural selection on bacteria resident in a eukaryotic host, a synthetic endosymbiont model system has been exploited. Here we describe a reproducible series of mutations that were observed after Escherichia coli was passaged within Saccharomyces cerevisiae for >8 passages which led to enhanced coexistence of bacteria within the yeast. These naturally selected mutations, formed by gene acquisition of trans-posable elements in rcsC, cpxA, and idnK, result in both functional and non-functional protein products with phenotypic effects on the bacteria that promote endosymbiont stability.
{"title":"Natural Selection in a Synthetic Yeast Endosymbiont Promotes Stable Coexistence","authors":"Lubica Supekova, Han Zhou, Izabella H. Barcellos, Catherine Nguyen, David A. Dik, Peter G. Schultz","doi":"10.1101/2024.07.31.606091","DOIUrl":"https://doi.org/10.1101/2024.07.31.606091","url":null,"abstract":"Bacteria engulfment by a higher order host is believed to be the beginning of an evolutionary process that ultimately formed mitochondria. In an effort to experimentally elucidate the early effects of natural selection on bacteria resident in a eukaryotic host, a synthetic endosymbiont model system has been exploited. Here we describe a reproducible series of mutations that were observed after <em>Escherichia coli</em> was passaged within <em>Saccharomyces cerevisiae</em> for >8 passages which led to enhanced coexistence of bacteria within the yeast. These naturally selected mutations, formed by gene acquisition of trans-posable elements in <em>rcsC, cpxA</em>, and <em>idnK</em>, result in both functional and non-functional protein products with phenotypic effects on the bacteria that promote endosymbiont stability.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"187 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141883763","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-31DOI: 10.1101/2024.07.31.605983
Armin M. Zand, Ankit Gupta, Mustafa Khammash
Precise intracellular regulation and robust perfect adaptation can be achieved using biomolecular integral controllers and it holds enormous potential for synthetic biology applications. In this letter, we consider the cascaded implementation of a class of such integrator motifs. Our cascaded integrators underpin proportional-integral-derivative (PID) control structures, which we leverage to suggest ways to improve dynamic performance. Moreover, we demonstrate how our cascaded strategy can be harnessed to enhance robust stability in a class of uncertain reaction networks. We also discuss the genetic implementation of our controllers and the natural occurrence of their cascaded sequestration pairs in bacterial pathogens.
{"title":"Cascaded Antithetic Integral Feedback Motifs for Robust Stability and Performance Improvement","authors":"Armin M. Zand, Ankit Gupta, Mustafa Khammash","doi":"10.1101/2024.07.31.605983","DOIUrl":"https://doi.org/10.1101/2024.07.31.605983","url":null,"abstract":"Precise intracellular regulation and robust perfect adaptation can be achieved using biomolecular integral controllers and it holds enormous potential for synthetic biology applications. In this letter, we consider the cascaded implementation of a class of such integrator motifs. Our cascaded integrators underpin proportional-integral-derivative (PID) control structures, which we leverage to suggest ways to improve dynamic performance. Moreover, we demonstrate how our cascaded strategy can be harnessed to enhance robust stability in a class of uncertain reaction networks. We also discuss the genetic implementation of our controllers and the natural occurrence of their cascaded sequestration pairs in bacterial pathogens.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"54 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141883765","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-07-30DOI: 10.1101/2024.07.30.604783
Angelica Frusteri Chiacchiera, Michela Casanova, Massimo Bellato, Aurora Piazza, Roberta Migliavacca, Gregory Batt, Paolo Magni, Lorenzo Pasotti
The global race against antimicrobial resistance requires novel antimicrobials that are not only effective in killing specific bacteria, but also minimize the emergence of new resistances. Recently, CRISPR/Cas-based antimicrobials were proposed to address killing specificity with encouraging results. However, the emergence of target sequence mutations triggered by Cas-cleavage was identified as an escape strategy, posing the risk of generating new antibiotic-resistance gene (ARG) variants. Here, we evaluated an antibiotic re-sensitization strategy based on CRISPR interference (CRISPRi), which inhibits gene expression without damaging target DNA. The resistance to four antibiotics, including last resort drugs, was significantly reduced by individual and multi-gene targeting of ARGs in low- to high-copy numbers in recombinant E. coli. Escaper analysis confirmed the absence of mutations in target sequence, corroborating the harmless role of CRISPRi in the selection of new resistances. E. coli clinical isolates carrying ARGs of severe clinical concern were then used to test the robustness of CRISPRi under different growth conditions. Meropenem, colistin and cefotaxime susceptibility was successfully increased in terms of MIC (up to >4-fold) and growth delay (up to 11-hours) in a medium-dependent fashion. To our knowledge, this is the first demonstration of CRISPRi-mediated re-sensitization to last-resort drugs in clinical isolates. This study laid the foundations for further leveraging CRISPRi as antimicrobial agent or research tool to selectively repress ARGs and investigate resistance mechanisms.
{"title":"Harnessing CRISPR interference to re-sensitize laboratory strains and clinical isolates to last resort antibiotics","authors":"Angelica Frusteri Chiacchiera, Michela Casanova, Massimo Bellato, Aurora Piazza, Roberta Migliavacca, Gregory Batt, Paolo Magni, Lorenzo Pasotti","doi":"10.1101/2024.07.30.604783","DOIUrl":"https://doi.org/10.1101/2024.07.30.604783","url":null,"abstract":"The global race against antimicrobial resistance requires novel antimicrobials that are not only effective in killing specific bacteria, but also minimize the emergence of new resistances. Recently, CRISPR/Cas-based antimicrobials were proposed to address killing specificity with encouraging results. However, the emergence of target sequence mutations triggered by Cas-cleavage was identified as an escape strategy, posing the risk of generating new antibiotic-resistance gene (ARG) variants. Here, we evaluated an antibiotic re-sensitization strategy based on CRISPR interference (CRISPRi), which inhibits gene expression without damaging target DNA. The resistance to four antibiotics, including last resort drugs, was significantly reduced by individual and multi-gene targeting of ARGs in low- to high-copy numbers in recombinant E. coli. Escaper analysis confirmed the absence of mutations in target sequence, corroborating the harmless role of CRISPRi in the selection of new resistances. E. coli clinical isolates carrying ARGs of severe clinical concern were then used to test the robustness of CRISPRi under different growth conditions. Meropenem, colistin and cefotaxime susceptibility was successfully increased in terms of MIC (up to >4-fold) and growth delay (up to 11-hours) in a medium-dependent fashion. To our knowledge, this is the first demonstration of CRISPRi-mediated re-sensitization to last-resort drugs in clinical isolates. This study laid the foundations for further leveraging CRISPRi as antimicrobial agent or research tool to selectively repress ARGs and investigate resistance mechanisms.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"41 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141872775","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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