Search for: All records

Abstract CRISPR ribonucleoproteins (RNPs) use a variable segment in their guide RNA (gRNA) called a spacer to determine the DNA sequence at which the effector protein will exhibit nuclease activity and generate target‐specific genetic mutations. However, nuclease activity with different gRNAs can vary considerably in a spacer sequence‐dependent manner that can be difficult to predict. While computational tools are helpful in predicting a CRISPR effector's activity and/or potential for off‐target mutagenesis with different gRNAs, individual gRNAs must still be validated in vitro prior to their use. Here, the study presents compartmentalized CRISPR reactions (CCR) for screening large numbers of spacer/target/off‐target combinations simultaneously in vitro for both CRISPR effector activity and specificity by confining the complete CRISPR reaction of gRNA transcription, RNP formation, and CRISPR target cleavage within individual water‐in‐oil microemulsions. With CCR, large numbers of the candidate gRNAs (output by computational design tools) can be immediately validated in parallel, and the study shows that CCR can be used to screen hundreds of thousands of extended gRNA (x‐gRNAs) variants that can completely block cleavage at off‐target sequences while maintaining high levels of on‐target activity. It is expected that CCR can help to streamline the gRNA generation and validation processes for applications in biological and biomedical research. 

Abstract Microfluidic valves play a key role within microfluidic systems by regulating fluid flow through distinct microchannels, enabling many advanced applications in medical diagnostics, lab‐on‐chips, and laboratory automation. While microfluidic systems are often limited to planar structures, 3D printing enables new capabilities to generate complex designs for fluidic circuits with higher densities and integrated components. However, the control of fluids within 3D structures presents several difficulties, making it challenging to scale effectively and many fluidic devices are still often restricted to quasi‐planar structures. Incorporating mechanical metamaterials that exhibit spatially adjustable mechanical properties into microfluidic systems provides an opportunity to address these challenges. Here, systematic computational and experimental characterization of a modified re‐entrant honeycomb structure are performed to generate a modular metamaterial for an active device that allows us to directly regulate flow through integrated, multiplexed fluidic channels “one‐at‐a‐time,” in a manner that is highly scalable. A design algorithm is presented, so that this architecture can be extended to arbitrary geometries, and it is expected that by incorporation of mechanical metamaterial designs into 3D printed fluidic systems, which themselves are readily expandable to any complex geometries, will enable new biotechnological and biomedical applications of 3D printed devices.