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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 While the archival digital memory industry approaches its physical limits, the demand is significantly increasing, therefore alternatives emerge. Recent efforts have demonstrated DNA’s enormous potential as a digital storage medium with superior information durability, capacity, and energy consumption. However, the majority of the proposed systems require on-demand de-novo DNA synthesis techniques that produce a large amount of toxic waste and therefore are not industrially scalable and environmentally friendly. Inspired by the architecture of semiconductor memory devices and recent developments in gene editing, we created a molecular digital data storage system called “DNA Mutational Overwriting Storage” (DMOS) that stores information by leveraging combinatorial, addressable, orthogonal, and independent in vitro CRISPR base-editing reactions to write data on a blank pool of greenly synthesized DNA tapes. As a proof of concept, this work illustrates writing and accurately reading of both a bitmap representation of our school’s logo and the title of this study on the DNA tapes. 

Abstract Solid surfaces that are immobilized with DNA molecules underlie an array of biotechnological devices. These surfaces may also mediate the self‐assembly of hierarchical DNA nanostructures. However, a number of fundamental questions concerning the structure–function relationship of these biointerfaces remain, including how these DNA probe molecules organize on the surface and how the spatial organization influences molecular recognition kinetics and interfacial affinity of these DNA molecules at the regime where crowding interactions are important (1–10 nm). This mini‐review covers recent advances in understanding this structure–function relationship by spatially resolving surface hybridization events at the single‐molecule level. Counterintuitive cooperative effects in surface hybridization are discussed and as is how modeling these cooperative effects can be used to predict the hybridization kinetics of a prototypical DNA sensor. Future opportunities in using mechanistic understanding to improve the performance and reliability of DNA sensors and form hierarchical supramolecular structures are also discussed.