Developing, characterizing and modeling CRISPR-based point-of-use pathogen diagnostics.

Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20 - 200 aM sensitivity without any specialized equipment. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

High-Throughput Regulatory Part Prototyping and Analysis by Cell-Free Protein Synthesis and Droplet Microfluidics.

Engineering regulatory parts for improved performance in genetic programs has played a pivotal role in the development of the synthetic biology cell programming toolbox. Here, we report the development of a novel high-throughput platform for regulatory part prototyping and analysis that leverages the advantages of engineered DNA libraries, cell-free protein synthesis (CFPS), high-throughput emulsion droplet microfluidics, standard flow sorting adapted to screen droplet reactions, and next-generation sequencing (NGS). With this integrated platform, we screened the activity of millions of genetic parts within hours, followed by NGS retrieval of the improved designs. This in vitro platform is particularly valuable for engineering regulatory parts of nonmodel organisms, where in vivo high-throughput screening methods are not readily available. The platform can be extended to multipart screening of complete genetic programs to optimize yield and stability.

Cell-Free Exploration of the Natural Product Chemical Space.

Natural products and secondary metabolites comprise an indispensable resource from living organisms that have transformed areas of medicine, agriculture, and biotechnology. Recent advances in high-throughput DNA sequencing and computational analysis suggest that the vast majority of natural products remain undiscovered. To accelerate the natural product discovery pipeline, cell-free metabolic engineering approaches used to develop robust catalytic networks are being repurposed to access new chemical scaffolds, and new enzymes capable of performing diverse chemistries. Such enzymes could serve as flexible biocatalytic tools to further expand the unique chemical space of natural products and secondary metabolites, and provide a more sustainable route to manufacture these molecules. Herein, we highlight select examples of natural product biosynthesis using cell-free systems and propose how cell-free technologies could facilitate our ability to access and modify these structures to transform synthetic and chemical biology.

Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins.

In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over polymerase chain reaction-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a double stranded DNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based protection of LET) method effectively increases superfolder green fluorescent protein (sfGFP) expression levels from LETs in Escherichia coli CFPS reactions by sixfold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from nonmodel organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.

Subcellular Control over Focal Adhesion Anisotropy, Independent of Cell Morphology, Dictates Stem Cell Fate.

Although microscale patterning techniques have been used to control cell morphology and shape, they only provide indirect control over the formation of the subcellular cytoskeletal elements that determine contractility. This paper addresses the hypotheses that nanoscale anisotropic features of a patterned matrix can direct the alignment of internal cytoskeletal actin fibers within a confined shape with an unbiased aspect ratio, and that this enhanced control over cytoskeletal architecture directs programmed cell behaviors. Here, large-area polymer pen lithography is used to pattern substrates with nanoscale extracellular matrix protein features and to identify cues that can be used to direct cytoskeletal organization in human mesenchymal stem cells. This nanopatterning approach is used to identify how anisotropic focal adhesions around the periphery of symmetric patterns yield an organized and contractile actin cytoskeleton. This work reports the important finding that anisotropic cues that increase cell contractility within a circular shape redirect cell differentiation from an adipogenic to an osteogenic fate. Together, these experiments introduce a programmable approach for using subcellular spatial cues to control cell behavior within defined geometries.

Nanopatterned Extracellular Matrices Enable Cell-Based Assays with a Mass Spectrometric Readout.

Cell-based assays are finding wider use in evaluating compounds in primary screens for drug development, yet it is still challenging to measure enzymatic activities as an end point in a cell-based assay. This paper reports a strategy that combines state-of-the-art cantilever free polymer pen lithography (PPL) with self-assembled monolayer laser desorption-ionization (SAMDI) mass spectrometry to guide cell localization and measure cellular enzymatic activities. Experiments are conducted with a 384 spot array, in which each spot is composed of ∼400 nanoarrays and each array has a 10 × 10 arrangement of 750 nm features that present extracellular matrix (ECM) proteins surrounded by an immobilized phosphopeptide. Cells attach to the individual nanoarrays, where they can be cultured and treated with small molecules, after which the media is removed and the cells are lysed. Phosphatase enzymes in the proximal lysate can then act on the immobilized phosphopeptide substrate to convert it to the dephosphorylated form. After the lysate is removed, the array is analyzed by SAMDI mass spectrometry to identify the extent of dephosphorylation and, therefore, the amount of enzyme activity in the cell. This novel approach of using nanopatterning to mediate cell adhesion and SAMDI to record enzyme activities in the proximal lysate will enable a broad range of cellular assays for applications in drug discovery and research not possible with conventional strategies.

Cell-Based Screening: Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry (Small 28/2016).

On page 3811, M. Mrksich and co-workers culture cells using self-assembled monolayers presenting cell adhesion ligands and enzyme substrates. A lysis buffer disrupts the cell membranes, releasing enzymes that modify the immobilized substrates. These modifications can be measured with SAMDI mass spectrometry, giving a high-throughput, cell-based assay.

Cellular Assays with a Molecular Endpoint Measured by SAMDI Mass Spectrometry.

Cell-based, high-throughput screening (HTS) assays are increasingly important tools used in drug discovery, but frequently rely on readouts of gene expression or phenotypic changes and require development of specialized, labeled reporters. Here a cell-based, label-free assay compatible with HTS is introduced that can report quantitatively on enzyme activities by measuring mass changes of substrates with matrix-assisted laser desorption/ionization mass spectrometry. The assay uses self-assembled monolayers to culture cells on arrays presenting substrates, which serve as reporters for a desired enzyme activity. Each spot of cells is treated with a compound, cultured and lysed, enabling endogenous enzymes to act on the immobilized peptide substrate. It is demonstrated that the assay can measure protein tyrosine phosphatase (PTP) activity from as few as five cells and a screen is described that identifies a compound that reduces PTP activity in cell lysates. This approach offers a valuable addition to the methods available for cell-based screening.

Hard Transparent Arrays for Polymer Pen Lithography.

Patterning nanoscale features across macroscopic areas is challenging due to the vast range of length scales that must be addressed. With polymer pen lithography, arrays of thousands of elastomeric pyramidal pens can be used to write features across centimeter-scales, but deformation of the soft pens limits resolution and minimum feature pitch, especially with polymeric inks. Here, we show that by coating polymer pen arrays with a ∼175 nm silica layer, the resulting hard transparent arrays exhibit a force-independent contact area that improves their patterning capability by reducing the minimum feature size (∼40 nm), minimum feature pitch (<200 nm for polymers), and pen to pen variation. With these new arrays, patterns with as many as 5.9 billion features in a 14.5 cm(2) area were written using a four hundred thousand pyramid pen array. Furthermore, a new method is demonstrated for patterning macroscopic feature size gradients that vary in feature diameter by a factor of 4. Ultimately, this form of polymer pen lithography allows for patterning with the resolution of dip-pen nanolithography across centimeter scales using simple and inexpensive pen arrays. The high resolution and density afforded by this technique position it as a broad-based discovery tool for the field of nanocombinatorics.

Nucleic acid-metal organic framework (MOF) nanoparticle conjugates.

Nanoparticles of a metal-organic framework (MOF), UiO-66-N3 (Zr6O4OH4(C8H3O4-N3)6), were synthesized. The surface of the MOF was covalently functionalized with oligonucleotides, utilizing a strain promoted click reaction between DNA appended with dibenzylcyclooctyne and azide-functionalized UiO-66-N3 to create the first MOF nanoparticle-nucleic acid conjugates. The structure of the framework was preserved throughout the chemical transformation, and the surface coverage of DNA was quantified. Due to the small pore sizes, the particles are only modified on their surfaces. When dispersed in aqueous NaCl, they exhibit increased stability and enhanced cellular uptake when compared with unfunctionalized MOF particles of comparable size.

Combinatorial screening of mesenchymal stem cell adhesion and differentiation using polymer pen lithography.

The extracellular matrix (ECM) is a complex, spatially inhomogeneous environment that is host to myriad cell-receptor interactions that promote changes in cell behavior. These biological systems can be probed and simulated with engineered surfaces, but doing so demands careful control over the arrangement of ligands. Here, we describe how such surfaces can be fabricated by utilizing polymer pen lithography (PPL), which is a cantilever-free scanning probe lithographic method that utilizes polymeric pen arrays to generate patterns over large areas. With the advent of PPL, fundamental questions in cell biology can be answered by recapitulating cell-ECM interactions to explore how these interactions lead to changes in cell behavior. Here, we describe an approach for the combinatorial screening of cell adhesion behavior to gain understanding of how ECM protein feature size dictates osteogenic differentiation of mesenchymal stem cells. The technique outlined here is generalizable to other biological systems and can be paired with quantitative analytical methods to probe important processes such as cell polarization, proliferation, signaling, and differentiation.

Large-area molecular patterning with polymer pen lithography.

The challenge of constructing surfaces with nanostructured chemical functionality is central to many areas of biology and biotechnology. This protocol describes the steps required for performing molecular printing using polymer pen lithography (PPL), a cantilever-free scanning probe-based technique that can generate sub-100-nm molecular features in a massively parallel fashion. To illustrate how such molecular printing can be used for a variety of biologically relevant applications, we detail the fabrication of the lithographic apparatus and the deposition of two materials, an alkanethiol and a polymer onto a gold and silicon surface, respectively, and show how the present approach can be used to generate nanostructures composed of proteins and metals. Finally, we describe how PPL enables researchers to easily create combinatorial arrays of nanostructures, a powerful approach for high-throughput screening. A typical protocol for fabricating PPL arrays and printing with the arrays takes 48-72 h to complete, including two overnight waiting steps.