Gene transcription (TX) and translation (TL) is executed is a single reaction tube with this highly efficient E.coli-based cell-free system.
Gene-editing technologies, including the widespread usage of CRISPR endonucleases, have the potential for clinical treatments of various human diseases. Due to the rapid mutations of SARS-CoV-2, specific and effective prevention and treatment by CRISPR toolkits for coronavirus disease 2019 (COVID-19) are urgently needed to control the current pandemic spread. Here, we designed Type III CRISPR endonuclease antivirals for coronaviruses (TEAR-CoV) as a therapeutic to combat SARS-CoV-2 infection. We provided a proof of principle demonstration that TEAR-CoV-based RNA engineering approach leads to RNA-guided transcript degradation both in vitro and in eukaryotic cells, which could be used to broadly target RNA viruses. We report that TEAR-CoV not only cleaves SARS-CoV-2 genome and mRNA transcripts, but also degrades live influenza A virus (IAV), impeding viral replication in cells and in mice. Moreover, bioinformatics screening of gRNAs along RNA sequences reveals that a group of five gRNAs (hCoV-gRNAs) could potentially target 99.98% of human coronaviruses. TEAR-CoV also exerted specific targeting and cleavage of common human coronaviruses. The fast design and broad targeting of TEAR-CoV may represent a versatile antiviral approach for SARS-CoV-2 or potentially other emerging human coronaviruses.
Protocol for preparation of chemo-competent KL740
cells for propagation of P70a vectors
In the ﬁght against antimicrobial resistance, bacteriophages are a promising alternative to antibiotics. However, due to their narrow spectra, phage therapy requires the careful matching between the host and bacteriophage to be eﬀective. Despite our best eﬀorts, nature remains as the only source of novel phage speciﬁcity. Directed evolution can potentially open an avenue for engineering phage speciﬁcity and improving qualities of phages that are not strongly selected for in their natural environments but are important for therapeutic applications. In this work, we present a strategy that generates large libraries of replication-competent phage variants directly from synthetic DNA fragments, with no restriction on their host speciﬁcity. Using the T7 bacteriophage as a proof-of-concept, we created a large library of tail ﬁber mutants with at least 107 unique variants. From this library, we identiﬁed mutants that have broadened speciﬁcity as evidenced by their novel lytic activity against Yersinia enterocolitica, a strain that the wildtype T7 was unable to lyse. Using the same concept, mutants with improved lytic eﬃciency and characteristics, such as lytic condition tolerance and resistance suppression, were also identiﬁed. However, the observed limitations in altering host speciﬁcity by tail ﬁber mutagenesis suggest that other bottlenecks could be of equal or even greater importance.
Cell-free transcription-translation (TXTL) systems produce RNAs and proteins from added DNA. By coupling their production to a biochemical assay, these biomolecules can be rapidly and scalably characterized without the need for purification or cell culturing. Here, we describe how TXTL can be applied to characterize Cas13 nucleases from Type VI CRISPR-Cas systems. These nucleases employ guide RNAs to recognize complementary RNA targets, leading to the nonspecific collateral cleavage of nearby RNAs. In turn, RNA targeting by Cas13 has been exploited for numerous applications, including in vitro diagnostics, programmable gene silencing in eukaryotes, and sequence-specific antimicrobials. As part of the described method, we detail how to set up TXTL assays to measure on-target and collateral RNA cleavage by Cas13 as well as how to assay for putative anti-CRISPR proteins. Overall, the method should be useful for the characterization of Type VI CRISPR-Cas systems and their use in ranging applications.
Cellular lysates capable of transcription and translation have become valuable tools for prototyping genetic circuits, screening engineered functional parts, and producing biological components. Here we report that lysates derived from Yersinia pestis CO92− are functional and can utilize both the E. coli σ70 and the bacteriophage T7 promoter systems to produce green ﬂuorescent protein (GFP). Because of the natural lifestyle of Y. pestis, lysates were produced from cultures grown at 21 °C, 26 °C, and 37 °C to mimic the infection cycle. Regardless of the promoter system the GFP production from 37 °C was the most productive and the 26 °C lysate was the least. When reactions are initiated with 5 nM of DNA, the GFP output of the 37 °C lysate is comparable with the productivity of other non-E. coli systems. The data we present demonstrate that, without genetic modiﬁcation to enhance productivity, cell-free extracts from Y. pestis are functional and dependent on the temperature at which the bacterium was grown.
Many bacterial mechanisms for highly specific and sensitive detection of heavy metals and other hazards have been reengineered to serve as sensors. In some cases, these sensors have been implemented in cell-free expression systems, enabling easier design optimization and deployment in low-resource settings through lyophilization. Here, we apply the advantages of cell-free expression systems to optimize sensors based on three separate bacterial response mechanisms for arsenic, cadmium, and mercury. We achieved detection limits below the World Health Organization-recommended levels for arsenic and mercury and below the short-term US Military Exposure Guideline levels for all three. The optimization of each sensor was approached differently, leading to observations useful for the development of future sensors: (1) there can be a strong dependence of specificity on the particular cell-free expression system used, (2) tuning of relative concentrations of the sensing and reporter elements improves sensitivity, and (3) sensor performance can vary significantly with linear vs plasmid DNA. In addition, we show that simply combining DNA for the three sensors into a single reaction enables detection of each target heavy metal without any further optimization. This combined approach could lead to sensors that detect a range of hazards at once, such as a panel of water contaminants or all known variants of a target virus. For low-resource settings, such “all-hazard” sensors in a cheap, easy-to-use format could have high utility.
Pyelonephritis-associated pili (pap) enable migration of the uropathogenic Escherichia coli strain (UPEC) through the urinary tract. UPEC can switch between a stable ‘ON phase’ where the corresponding pap genes are expressed and a stable ‘OFF phase’ where their transcription is repressed. Hereditary DNA methylation of either one of two GATC motives within the regulatory region stabilizes the respective phase over many generations. The underlying molecular mechanism is only partly understood. Previous investigations suggest that in vivo phase-variation stability results from cooperative action of the transcriptional regulators Lrp and PapI. Here, we use an E. coli cell-free expression system to study molecular functions of the pap regulatory region based on a specially designed, synthetic construct ﬂanked by two reporter genes encoding ﬂuorescent proteins for simple readout. On the basis of our observations we suggest that besides Lrp, the conformation of the self-complementary regulatory DNA plays a strong role in the regulation of phase-variation. Our work not only contributes to better understand the phase variation mechanism, but it represents a successful start for mimicking stable, hereditary, and strong expression control based on methylation. The conformation of the regulatory DNA corresponds to a Holliday junction. Gene expression must be expected to respond if opposite arms of the junction are drawn outward.
A major goal of synthetic biology is to understand the transition between non-living matter and life. The bottom-up development of an artificial cell would provide a minimal system with which to study the border between chemistry and biology. So far, a fully synthetic cell has remained elusive, but chemists are progressing towards this goal by reconstructing cellular subsystems. Cell boundaries, likely in the form of lipid membranes, were necessary for the emergence of life. In addition to providing a protective barrier between cellular cargo and the external environment, lipid compartments maintain homeostasis with other subsystems to regulate cellular processes. In this Review, we examine different chemical approaches to making cell-mimetic compartments. Synthetic strategies to drive membrane formation and function, including bioorthogonal ligations, dissipative self-assembly and reconstitution of biochemical pathways, are discussed. Chemical strategies aim to recreate the interactions between lipid membranes, the external environment and internal biomolecules, and will clarify our understanding of life at the interface of chemistry and biology.
Cell-free protein synthesis (CFPS) is a platform biotechnology that has enabled the on-demand synthesis of proteins for a variety of applications. Numerous advances have improved the productivity of the CFPS platform to result in high-yielding reactions; however, many applications remain limited due to long reaction times. To overcome this limitation, we first established the benchmarks reaction times for CFPS across in-house E. coli extracts and commercial kits. We then set out to fine-tune our in-house extract systems to improve reaction times. Through the optimization of reaction composition and titration of low-cost additives, we have identified formulations that reduce reaction times by 30–50% to obtain high protein titers for biomanufacturing applications, and reduce times by more than 50% to reach the sfGFP detection limit for applications in education and diagnostics. Under optimum conditions, we report the visible observation of sfGFP signal in less than 10 min. Altogether, these advances enhance the utility of CFPS as a rapid, user-defined platform.
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