bioRxiv - Synthetic Biology最新文献_第6页

Drug-controlled CAR-T cells through the regulation of cell-cell interactions 通过调节细胞-细胞间的相互作用实现药物控制的 CAR-T 细胞

bioRxiv - Synthetic Biology Pub Date : 2024-08-06 DOI: 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

{"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":null,"pages":null},"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}

引用次数: 0

Colorimetric CRISPR Biosensor: A Case Study with Salmonella Typhi 比色 CRISPR 生物传感器：伤寒沙门氏菌案例研究

bioRxiv - Synthetic Biology Pub Date : 2024-08-05 DOI: 10.1101/2024.08.05.606709

Ana Pascual-Garrigos, Beatriz Lozano-Torres, Akashaditya Das, Jennifer C Molloy

{"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":null,"pages":null},"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}

引用次数: 0

Random Sanitization in DNA information storage using CRISPR-Cas12a 利用 CRISPR-Cas12a 对 DNA 信息存储进行随机消毒

bioRxiv - Synthetic Biology Pub Date : 2024-08-05 DOI: 10.1101/2024.08.04.606549

Hongyu Shen, Zhi Weng, Haipei Zhao, Haitao Song, Fei Wang, Chunhai Fan, Ping Song

{"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":null,"pages":null},"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}

引用次数: 0

Accelerated enzyme engineering by machine-learning guided cell-free expression 通过机器学习引导的无细胞表达加速酶工程

bioRxiv - Synthetic Biology Pub Date : 2024-08-02 DOI: 10.1101/2024.07.30.605672

Grant M Landwehr, Jonathan W Bogart, Carol Magalhaes, Eric Hammarlund, Ashty S Karim, Michael C Jewett

引用次数: 0

Establishment of a rapid method to assemble and transfer DNA fragments into the JCVI-syn3B minimal synthetic bacterial genome 建立将 DNA 片段组装和转移到 JCVI-syn3B 最小合成细菌基因组的快速方法

bioRxiv - Synthetic Biology Pub Date : 2024-08-01 DOI: 10.1101/2024.08.01.606163

Atsuko Uenoyama, Hana Kiyama, Mone Mimura, Makoto Miyata

引用次数: 0

moPPIt: De Novo Generation of Motif-Specific Binders with Protein Language Models moPPIt：利用蛋白质语言模型从新生成特定于动机的粘合剂

bioRxiv - Synthetic Biology Pub Date : 2024-08-01 DOI: 10.1101/2024.07.31.606098

Tong Chen, Yinuo Zhang, Pranam Chatterjee

{"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 motif-specific PPI targeting 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.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"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}

引用次数: 0

Delivery of novel replicating vectors to Synechococcus sp. PCC 7002 via natural transformation of plasmid multimers 通过质粒多聚体的自然转化向 Synechococcus sp.

bioRxiv - Synthetic Biology Pub Date : 2024-07-31 DOI: 10.1101/2024.07.31.606084

Cody Kamoku, Cheyanna Cooper, Ashley Straub, Nathan Miller, David R Nielsen

{"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 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.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":null,"pages":null},"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}

引用次数: 0

Natural Selection in a Synthetic Yeast Endosymbiont Promotes Stable Coexistence 合成酵母内共生体的自然选择促进稳定共存

bioRxiv - Synthetic Biology Pub Date : 2024-07-31 DOI: 10.1101/2024.07.31.606091

Lubica Supekova, Han Zhou, Izabella H. Barcellos, Catherine Nguyen, David A. Dik, Peter G. Schultz

引用次数: 0

Cascaded Antithetic Integral Feedback Motifs for Robust Stability and Performance Improvement 级联式反嵌合积分反馈动机实现稳健稳定性和性能改进

bioRxiv - Synthetic Biology Pub Date : 2024-07-31 DOI: 10.1101/2024.07.31.605983

Armin M. Zand, Ankit Gupta, Mustafa Khammash

引用次数: 0

Harnessing CRISPR interference to re-sensitize laboratory strains and clinical isolates to last resort antibiotics 利用 CRISPR 干扰使实验室菌株和临床分离菌株对最后的抗生素重新敏感

bioRxiv - Synthetic Biology Pub Date : 2024-07-30 DOI: 10.1101/2024.07.30.604783

Angelica Frusteri Chiacchiera, Michela Casanova, Massimo Bellato, Aurora Piazza, Roberta Migliavacca, Gregory Batt, Paolo Magni, Lorenzo Pasotti

{"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":null,"pages":null},"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}

引用次数: 0