The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP.
Gene therapy has long held promise to correct a variety of human diseases and defects. Discovery of the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR), the mechanism of the CRISPRbased prokaryotic adaptive immune system (CRISPR-associated system, Cas), and its repurposing into a potent gene editing tool has revolutionized the field of molecular biology and generated excitement for new and improved gene therapies. Additionally, the simplicity and flexibility of the CRISPR/Cas9 site-specific nuclease system has led to its widespread use in many biological research areas including development of model cell lines, discovering mechanisms of disease, identifying disease targets, development of transgene animals and plants, and transcriptional modulation. In this review, we present the brief history and basic mechanisms of the CRISPR/Cas9 system and its predecessors (ZFNs and TALENs), lessons learned from past human gene therapy efforts, and recent modifications of CRISPR/ Cas9 to provide functions beyond gene editing. We introduce several factors that influence CRISPR/ Cas9 efficacy which must be addressed before effective in vivo human gene therapy can be realized. The focus then turns to the most difficult barrier to potential in vivo use of CRISPR/Cas9, delivery. We detail the various cargos and delivery vehicles reported for CRISPR/Cas9, including physical delivery methods (e.g. microinjection; electroporation), viral delivery methods (e.g. adeno-associated virus (AAV); full-sized adenovirus and lentivirus), and non-viral delivery methods (e.g. liposomes; polyplexes; gold particles), and discuss their relative merits. We also examine several technologies that, while not currently reported for CRISPR/Cas9 delivery, appear to have promise in this field. The therapeutic potential of CRISPR/Cas9 is vast and will only increase as the technology and its delivery improves.
Over the past 20 years, many strategies utilizing sol-gel chemistry to integrate biological cells into silica-based materials have been reported. One such strategy, Sol-Generating Chemical Vapor into Liquid (SG-CViL) deposition, shows promise as an efficient encapsulation technique due to the ability to vary the silica encapsulation morphology obtained by this process through variation of SG-CViL reaction conditions. In this report, we develop SG-CViL as a tunable, multi-purpose silica encapsulation strategy by investigating the mechanisms governing both silica particle generation and subsequent interaction with phospholipid assemblies (liposomes and living cells). Using Dynamic Light Scattering (DLS) measurements, linear and exponential silica particle growth dynamics were observed which were dependent on deposition buffer ion constituents and ion concentration. Silica particle growth followed a cluster-cluster growth mechanism at acidic pH, and a monomer-cluster growth mechanism at neutral to basic pH. Increasing silica sol aging temperature resulted in higher rates of particle growth and larger particles. DLS measurements employing PEG-coated liposomes and cationic liposomes, serving as model phospholipid assemblies, revealed that electrostatic interactions promote more stable liposome-silica interactions than hydrogen bonding and facilitate silica coating on suspension cells. However, continued silica reactivity leads to aggregation of silica-coated suspension cells, revealing the need for cell isolation to tune deposited silica thickness. Utilizing these mechanistic study insights, silica was deposited onto adherent HeLa cells under biocompatible conditions with micrometer-scale control over silica thickness, minimal cell manipulation steps, and retained cell viability over several days.
The remarkable impact encapsulation matrix chemistry can have on the bioactivity and viability of integrated living cells is reported. Two silica chemistries (aqueous silicate and alkoxysilane), and a functional component additive (glycerol), are employed to generate three distinct silica matrices. These matrices are used to encapsulate living E. coli cells engineered with a synthetic riboswitch for cell-based biosensing. Following encapsulation, membrane integrity, reproductive capability, and riboswitch-based protein expression levels and rates are measured over a 5 week period. Striking differences in E. coli bioactivity, viability, and biosensing performance are observed for cells encapsulated within the different matrices. E. coli cells encapsulated for 35 days in aqueous silicate-based (AqS) matrices showed relatively low membrane integrity, but high reproductive capability in comparison to cells encapsulated in glycerol containing sodium silicate-based (AqS + g) and alkoxysilane-based (PGS) gels. Further, cells in sodium silicate-based matrices showed increasing fluorescence output over time, resulting in a 1.8-fold higher fluorescence level, and a faster expression rate, over cells free in solution. This unusual and unique combination of biological properties demonstrates that careful design of the encapsulation matrix chemistry can improve functionality of the biocomposite material, and result in new and unexpected physiological states.
Despite significant progress in development of bioanalytical devices cost, complexity, access to reagents and lack of infrastructure have prevented use of these technologies in resource-limited regions. To provide a sustainable tool in the global effort to combat infectious diseases the diagnostic device must be low cost, simple to operate and read, robust, and have sensitivity and specificity comparable to laboratory analysis. In this mini-review we describe recent work using laser machined plastic laminates to produce diagnostic devices that are capable of a wide variety of bioanalytical measurements and show great promise towards future use in low-resource environments.
Johnston, Robert; Rogelj, Snezna; Harper, Jason C.; Tartis, Michaelann
In nature, cells perform a variety of complex functions such as sensing, catalysis, and energy conversion which hold great potential for biotechnological device construction. However, cellular sensitivity to ex vivo environments necessitates development of bio-nano interfaces which allow integration of cells into devices and maintain their desired functionality. In order to develop such an interface, the use of a novel Sol-Generating Chemical Vapor into Liquid (SG-CViL) deposition process for whole cell encapsulation in silica was explored. In SG-CViL, the high vapor pressure of tetramethyl orthosilicate (TMOS) is utilized to deliver silica into an aqueous medium, creating a silica sol. Cells are then mixed with the resulting silica sol, facilitating encapsulation of cells in silica while minimizing cell contact with the cytotoxic products of silica generating reactions (i.e. methanol), and reduce exposure of cells to compressive stresses induced from silica condensation reactions. Using SG-CVIL, Saccharomyces cerevisiae (S. cerevisiae) engineered with an inducible beta galactosidase system were encapsulated in silica solids and remained both viable and responsive 29 days post encapsulation. By tuning SG-CViL parameters, thin layer silica deposition on mammalian HeLa and U87 human cancer cells was also achieved. The ability to encapsulate various cell types in either a multi cell (S. cerevisiae) or a thin layer (HeLa and U87 cells) fashion shows the promise of SG-CViL as an encapsulation strategy for generating cell-silica constructs with diverse functions for incorporation into devices for sensing, bioelectronics, biocatalysis, and biofuel applications.