CRISPR-Cas System: The Current and Emerging Translational Landscape.

CRISPR-Cas technology has rapidly changed life science research and human medicine. The ability to add, remove, or edit human DNA sequences has transformative potential for treating congenital and acquired human diseases. The timely maturation of the cell and gene therapy ecosystem and its seamless integration with CRISPR-Cas technologies has enabled the development of therapies that could potentially cure not only monogenic diseases such as sickle cell anemia and muscular dystrophy, but also complex heterogenous diseases such as cancer and diabetes. Here, we review the current landscape of clinical trials involving the use of various CRISPR-Cas systems as therapeutics for human diseases, discuss challenges, and explore new CRISPR-Cas-based tools such as base editing, prime editing, CRISPR-based transcriptional regulation, CRISPR-based epigenome editing, and RNA editing, each promising new functionality and broadening therapeutic potential. Finally, we discuss how the CRISPR-Cas system is being used to understand the biology of human diseases through the generation of large animal disease models used for preclinical testing of emerging therapeutics.

Transglutaminase-mediated assembly of multi-enzyme pathway onto TMV brush surfaces for synthesis of bacterial autoinducer-2.

Bioelectronic microdevices, with spatially arranged biosynthetic machinery, can be programmed to convert raw materials to high-value products in a controlled manner. Generic methods for biofunctionalization that enable precise control over biocomponent assembly at the nano and meso scales are necessary to diversify the range and capabilities of these systems. Here, we used tobacco mosaic virus (TMV) derived virus like particles (VLPs) as 3D interfacial scaffolds for the assembly of biosynthetic enzymes onto gold electrodes. The TMV capsids are aligned in a vertical brush configuration by cysteine modifications to the capsid protein and by taking advantage of the well-known gold/cysteine affinity. This alignment enables high surface density and biosynthetic enzyme-enzyme proximity. Enzymes are covalently tethered to the capsid protein of TMV by the N- and C-terminal addition of lysine-rich assembly domains which react with surface exposed glutamine residues on the capsid surfaces; the lysine/glutamine linkages are mediated by a microbial transglutaminase (mTG). We demonstrate flexible mTG-mediated assembly of a three-enzyme biosynthetic pathway that converts S-adenosylmethionine (SAM) to autoinducer-2 (AI-2), a bacterial signal molecule that mediates quorum sensing behavior. We propose that our VLP and mTG based fabrication approach will help in the modular assembly of biological components onto microelectronic devices and that these will find utility in many applications including sensing and lab on chip devices.

A redox-based electrogenetic CRISPR system to connect with and control biological information networks.

Electronic information can be transmitted to cells directly from microelectronics via electrode-activated redox mediators. These transmissions are decoded by redox-responsive promoters which enable user-specified control over biological function. Here, we build on this redox communication modality by establishing an electronic eCRISPR conduit of information exchange. This system acts as a biological signal processor, amplifying signal reception and filtering biological noise. We electronically amplify bacterial quorum sensing (QS) signaling by activating LasI, the autoinducer-1 synthase. Similarly, we filter out unintended noise by inhibiting the native SoxRS-mediated oxidative stress response regulon. We then construct an eCRISPR based redox conduit in both E. coli and Salmonella enterica. Finally, we display eCRISPR based information processing that allows transmission of spatiotemporal redox commands which are then decoded by gelatin-encapsulated E. coli. We anticipate that redox communication channels will enable biohybrid microelectronic devices that could transform our abilities to electronically interpret and control biological function.

Catechol-chitosan redox capacitor for added amplification in electrochemical immunoanalysis.

Antibodies are common recognition elements for molecular detection but often the signals generated by their stoichiometric binding must be amplified to enhance sensitivity. Here, we report that an electrode coated with a catechol-chitosan redox capacitor can amplify the electrochemical signal generated from an alkaline phosphatase (AP) linked immunoassay. Specifically, the AP product p-aminophenol (PAP) undergoes redox-cycling in the redox capacitor to generate amplified oxidation currents. We estimate an 8-fold amplification associated with this redox-cycling in the capacitor (compared to detection by a bare electrode). Importantly, this capacitor-based amplification is generic and can be coupled to existing amplification approaches based on enzyme-linked catalysis or magnetic nanoparticle-based collection/concentration. Thus, the capacitor should enhance sensitivities in conventional immunoassays and also provide chemical to electrical signal transduction for emerging applications in molecular communication.

Biofabricating Functional Soft Matter Using Protein Engineering to Enable Enzymatic Assembly.

Biology often provides the inspiration for functional soft matter, but biology can do more: it can provide the raw materials and mechanisms for hierarchical assembly. Biology uses polymers to perform various functions, and biologically derived polymers can serve as sustainable, self-assembling, and high-performance materials platforms for life-science applications. Biology employs enzymes for site-specific reactions that are used to both disassemble and assemble biopolymers both to and from component parts. By exploiting protein engineering methodologies, proteins can be modified to make them more susceptible to biology's native enzymatic activities. They can be engineered with fusion tags that provide (short sequences of amino acids at the C- and/or N- termini) that provide the accessible residues for the assembling enzymes to recognize and react with. This "biobased" fabrication not only allows biology's nanoscale components (i.e., proteins) to be engineered, but also provides the means to organize these components into the hierarchical structures that are prevalent in life.

A Facile Two-Step Enzymatic Approach for Conjugating Proteins to Polysaccharide Chitosan at an Electrode Interface.

Biological components are integrated with electronic devices to create microsystems with novel functions and chitosan, a naturally occurring biopolymer, can play a significant role as an interface material. Chitosan can be electrodeposited within confined geometries by cathodic charge and appropriate electrode design and proteins can be conjugated to chitosan. However, conjugation chemistries can be slow and chitosan, a polycationic polysaccharide, enables non-specific binding in biofabrication processes. There is a need to speed up the assembly process and reduce non-specific binding. Here, we have developed a two-step methodology that accelerates protein assembly, reduces background and increases specificity. We first "coated" the surface of chitosan with a Lys-Tyr-Lys (KYK) tripeptide in a slow step using tyrosinase-mediated conjugation chemistry and then conjugated proteins with C-terminal glutamine-tags to the saturating KYK tripeptide via transglutaminase. As a demonstration, we assembled a functioning two-enzyme bacterial metabolic pathway on an electrode chip. Results indicated a fivefold decrease in non-specific binding and an improvement in signal to noise ratio from 0.3 to 20. This transglutaminase-mediated approach is simple and quick, it requires no chemical reagents, no printing or stamping devices; it employs biological components and is biologically benign to the component parts-all characteristics of biofabricated devices.

Data on biochemical fluxes generated from biofabricated enzyme complexes assembled through engineered tags and microbial transglutaminase.

Data presented is related to an article titled "Modular construction of multi-subunit protein complexes using engineered tags and microbial transglutaminase" (Bhokisham et al., 2016) [1]. In this article, we have presented western blot and flux data associated with assembly of Pfs-LuxS enzyme complexes on beads using uni-tagged and bi-tagged LuxS enzymes. We have also presented biochemical flux following changes in enzyme stoichiometries. We covalently coupled a Pfs-LuxS complex with Protein G, an antibody binding non-enzyme component and directed these complexes to the surfaces of bacterial cells via anti-Escherichia coli antibodies. Fluorescence microscopy images represented the altered behavior of bacterial cells in response to the autoinducer-2 that is synthesized by the Protein G-enzyme complexes.

Modular construction of multi-subunit protein complexes using engineered tags and microbial transglutaminase.

Motivations for the hierarchical assembly of protein complexes are diverse spanning biosensing, biomedical and bioreactor applications. The assembly processes should be simple, scalable, versatile, and biologically benign to minimize loss of component parts. A "plug and play" methodology comprising a generic linking apparatus may enable rapid design and optimization. One application that desires these qualities is metabolon construction wherein multiple enzymes are organized in defined pathways to mediate biochemical flux. Here, we propose a modular design by incorporation of crosslinking-compliant amino acid tags comprised of lysine or glutamine residues at the N- or C-termini of the to-be-assembled proteins. These amino acid tags enable covalent crosslinking using microbial transglutaminase (mTG). Modularity is demonstrated where stoichiometries and relative positions of enzymes and other functional proteins are altered. Construction of multifunctional complexes is demonstrated by crosslinking domains of different function and origin. Namely, we built a two-subunit quorum sensing (QS) biosynthetic metabolon on solid supports and altered stoichiometries of the limiting constituents to increase the overall rate of reaction. To display functionality beyond biosynthesis, we constructed a molecular communication 'device' (antibody binding Protein G-QS complex) to target bacterial cells and demonstrated tailored QS responses among targeted bacteria. We propose that this approach, solid phase mTG-mediated linkage of biological components, can be used for assembly within many environments including microreactors or lab-on-a-chip systems. Because the methodology is general, we envision construction of multi-functional protein complexes in a 'plug and play' fashion for a variety of biosensing and synthetic biology applications.