Abstract:Creating CRISPR-responsive smart materials for diagnostics and programmable cargo releaseThe MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters.
CitationGayet, Raphael V. et al. "Creating CRISPR-responsive smart materials for diagnostics and programmable cargo release." Nature Protocols 15, 9 (August 2020): 3030-3063.
“…Another possibility could be to engineer a companion device to automate reagent dispensing and regulate reaction temperature and timing. Prototypes for automated, sequential introduction of reagents 35, 36 and portable incubators 7 have been developed by different labs for point-of-care diagnostics. Though these additions would increase the capital cost of the equipment and the cost per assay, thus limiting its potential use in low-resource environments, it would still be sufficiently accessible for patient at-home use and clinic use in the developed world.…”
Field-deployable diagnostics based on cell-free systems have advanced greatly, but on-site quantification of target analytes remains a challenge. Here we demonstrate that Escherichia coli lysate-based cell-free biosensors coupled to a personal glucose monitor (PGM) can enable on-site analyte quantification, with the potential for straightforward reconfigurability to diverse types of analytes. We show that analyte-responsive regulators of transcription and translation can modulate production of the reporter enzyme β-galactosidase, which in turn converts lactose into glucose for PGM quantification. Because glycolysis is active in the lysate and would readily deplete converted glucose, we decoupled enzyme production and glucose conversion to increase endpoint signal output. This lysate metabolism did, however, allow for one-pot removal of glucose present in complex samples (like human serum) without confounding target quantification. Taken together, we show that integrating lysate-based cell-free biosensors with PGMs enables accessible target detection and quantification at the point of need.
“…Another possibility could be to engineer a companion device to automate reagent dispensing and regulate reaction temperature and timing. Prototypes for automated, sequential introduction of reagents 35, 36 and portable incubators 7 have been developed by different labs for point-of-care diagnostics. Though these additions would increase the capital cost of the equipment and the cost per assay, thus limiting its potential use in low-resource environments, it would still be sufficiently accessible for patient at-home use and clinic use in the developed world.…”
Field-deployable diagnostics based on cell-free systems have advanced greatly, but on-site quantification of target analytes remains a challenge. Here we demonstrate that Escherichia coli lysate-based cell-free biosensors coupled to a personal glucose monitor (PGM) can enable on-site analyte quantification, with the potential for straightforward reconfigurability to diverse types of analytes. We show that analyte-responsive regulators of transcription and translation can modulate production of the reporter enzyme β-galactosidase, which in turn converts lactose into glucose for PGM quantification. Because glycolysis is active in the lysate and would readily deplete converted glucose, we decoupled enzyme production and glucose conversion to increase endpoint signal output. This lysate metabolism did, however, allow for one-pot removal of glucose present in complex samples (like human serum) without confounding target quantification. Taken together, we show that integrating lysate-based cell-free biosensors with PGMs enables accessible target detection and quantification at the point of need.
“…The Collins group recently pioneered a new class of nucleic acid–responsive hydrogels by the development of the first CRISPR-Cas–responsive material ( Fig. 7 ) [ 170 , 171 ]. The system relied on Lachnospiraceae bacterium ND2006-derived Cas12a, that displays specific nuclease activity toward double-stranded DNA (dsDNA) sequences matching the gRNA spacer sequence, and subsequent non-specific hydrolase activity toward (ssDNA).…”
Section: Engineering Cells To Synthesize (Precursors Of) Non-living Materialsmentioning
confidence: 99%
“…The main advantage of these CRISPR-Cas–responsive gels is their flexibility. By simply exchanging the gRNA sequence, the materials can be engineered to specifically detect dsDNA sequences of choice [ 171 ]. Fig.…”
Section: Engineering Cells To Synthesize (Precursors Of) Non-living Materialsmentioning
confidence: 99%
“…Polyacrylamide functionalized with ssDNA sequences was crosslinked to a hydrogel by complementary ssDNA oligonucleotides. Addition of gRNA-loaded Cas12a and a cognate dsDNA induced non-specific ssDNA nuclease activity, the cleavage of the crosslinker ssDNA and the dissolution of the hydrogel [ 170 , 171 ]. …”
Section: Engineering Cells To Synthesize (Precursors Of) Non-living Materialsmentioning
Materials in nature have fascinating properties that serve as a continuous source of inspiration for materials scientists. Accordingly, bio-mimetic and bio-inspired approaches have yielded remarkable structural and functional materials for a plethora of applications. Despite these advances, many properties of natural materials remain challenging or yet impossible to incorporate into synthetic materials. Natural materials are produced by living cells, which sense and process environmental cues and conditions by means of signaling and genetic programs, thereby controlling the biosynthesis, remodeling, functionalization, or degradation of the natural material. In this context, synthetic biology offers unique opportunities in materials sciences by providing direct access to the rational engineering of how a cell senses and processes environmental information and translates them into the properties and functions of materials. Here, we identify and review two main directions by which synthetic biology can be harnessed to provide new impulses for the biologization of the materials sciences: first, the engineering of cells to produce precursors for the subsequent synthesis of materials. This includes materials that are otherwise produced from petrochemical resources, but also materials where the bio-produced substances contribute unique properties and functions not existing in traditional materials. Second, engineered living materials that are formed or assembled by cells or in which cells contribute specific functions while remaining an integral part of the living composite material. We finally provide a perspective of future scientific directions of this promising area of research and discuss science policy that would be required to support research and development in this field.
“…The CRISPR-Cas RNA detection experiments reported here capitalize on Cas12a from Lachnospiraceae bacterium ND2006, which has specific cleavage activity towards dsDNA fragments matching its guide RNA (gRNA) sequence. Upon target binding, activated Cas12a-gRNA engages in collateral cleavage of nearby single-stranded DNA (ssDNA) 17,18 which can be read optically as an increase in fluorescence due to the hydrolysis of a fluorophore-quencher labeled ssDNA reporter. LAMP primers [28][29][30][31][32] and Cas12a-gRNAs were evaluated from a range of conserved regions in the SARS-CoV-2 genome to determine the most sensitive combinations using commercially available, synthetic full-length SARS-CoV-2 genomic RNA.…”
Section: Optimization Of a Crispr-based Sensor For Sars-cov-2 Viral Rnamentioning
There continues to be a great need for rapid, accurate, and cost-effective point-of-care devices that can diagnose the presence of SARS-CoV-2 virus and development of IgG and IgM antibody responses in early and late stages of COVID-19 disease. Here, we describe a versatile multiplexed electrochemical (EC) sensor platform modified with an antifouling nanocomposite coating that enables single-molecule CRISPR/Cas-based molecular detection of SARS-CoV-2 viral RNA with on-chip signal validation as well as multiplexed serological detection of antibodies against three SARS-CoV-2 viral antigens. The CRISPR-based EC platform achieved 100% accuracy for detection of viral RNA and showed an excellent correlation with RT-qPCR using 30 clinical saliva samples. The serology EC platform obtained 100% sensitivity and 100% specificity for anti-SARS-CoV-2 IgG, as well as 94% specificity and 82% sensitivity for anti-SARS-CoV-2 IgM with 112 clinical plasma samples. These data demonstrate that integration of CRISPR-based RNA detection and serological assays with antifouling nanocomposite-based EC sensors enables performance as good or better than traditional laboratory-based techniques.
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