🧬 PubMed RNA Editing Daily Digest

CRISPR-Cas effectors typically rely on RNA guides to recognize target sequences. In Cas12a, the protospacer adjacent motif on DNA engages conserved protein residues, triggering target binding and nuclease activation. Here we reprogram Cas12a into a DNA-guided, RNA-targeting effector. Exploiting protospacer-adjacent motif-dependent interaction, we engineer synthetic CRISPR DNA that engages Cas12a to form a functional deoxyribonucleoprotein complex, while repurposing solely RNA as the programmable target. Structural, biophysical and biochemical analyses reveal the molecular basis of this DNA-guided, RNA-targeting configuration and support an activation pathway distinct from that of canonical RNA-guided systems. DNA-guided Cas12a enables direct RNA detection and efficient intracellular RNA knockdown, establishing a modular activation architecture for CRISPR-Cas12a and expanding the design space for programmable RNA manipulation.

Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous group of disorders affecting millions worldwide, with limited therapeutic options for the majority of patients. Recent advances in RNA editing-particularly adenosine-to-inosine (A-to-I) editing mediated by adenosine deaminases acting on RNA (ADAR)- offer a promising approach for correcting pathogenic variants at the RNA level without altering the genome. This review presents a comprehensive overview of the molecular basis of ADAR-mediated RNA editing, its natural occurrence in retinal tissues, and the growing array of strategies that harness endogenous ADAR enzymes for site-directed RNA editing (SDRE). We discuss design principles and optimization strategies for guide RNAs (gRNAs), including linear, circular, and chemically modified formats such as LEAPER, RESTORE, CLUSTER, CadRNAs, and AIMers. Additionally, we explore high-throughput screening systems for guide selection, cellular models for assessing RNA editing efficiency, and current in vitro and in vivo approaches targeting IRD pathogenic variants. Finally, we highlight the translational potential of endogenous and exogenous ADAR-based RNA editing, emphasizing its relevance as a precision medicine strategy for IRDs and beyond.

RNA naturally regulates many cellular processes, yet the engineering of RNA for use in synthetic cellular control schemes lags behind protein-based systems. Recent advancements in synthetic biology, investment in RNA therapeutics, and a better understanding of RNA structural dynamics have driven the development of novel RNA sensors and actuators. The genetic information encoded within RNA enables facile sensing and interactions with other nucleic acids, while its dynamic structure facilitates binding to a broad array of small-molecule and protein ligands. RNA can be engineered to sense these diverse inputs and transduce signals to regulate cellular activity on the transcriptional, translational, and post-translational levels to enhance microbial biosynthesis and create targeted gene therapies.

CRISPR-Cas technology has emerged as a transformative tool with widespread applications in gene editing and biosensing research; nevertheless, it is plagued by a suite of performance-related bottlenecks, including suboptimal targeting efficiency, undesirable off-target effects, insufficient sensitivity and recognition specificity, restricted target scope, limited multiplexing capacity, incompatible reaction systems, and compromised stability. As gRNA optimization has emerged as a core strategy to address these bottlenecks, there is an urgent need to consolidate recent breakthroughs in this rapidly advancing field. Existing literature lacks a comprehensive, focused synthesis of how gRNA optimization mitigates these key limitations, alongside an analysis of current challenges and future directions. Herein, this review comprehensively summarizes recent breakthroughs in augmenting CRISPR-Cas system performance through guide RNA (gRNA) optimization, and further dissects the current challenges, future prospects, and promising research directions in this rapidly advancing field. It is timely to guide researchers in overcoming CRISPR-Cas performance barriers and accelerating its applications in gene editing and biosensing.

Optical pooled CRISPR screens have become an attractive tool for the rapid identification of genes involved in biological processes. In such screens, mixed populations of cells, each with a single gene knocked out, are screened by microscopy for phenotypes of interest. Identified hit cells can then be tagged by photoactivation of a co-expressed marker, such as PA-mCherry, and subsequently isolated by FACS to identify the responsible guide RNA by next-generation sequencing. Photoactivation is typically performed by selective irradiation of cells with UV light, using either a digital mirror device (DMD), an external fixed UV laser, or, conveniently, by using the 405 nm laser line present in most confocal scanning microscopes. In this study, the latter approach is optimized for PA-mCherry, a bright red phototag used by us and others in optical pooled screens. We find that although normal scanning with intense 405 nm light can rapidly activate PA-mCherry, it also leads to rapid photobleaching. Instead, much higher cellular brightness is achieved by limiting intensity and pixel dwell time during scanning, as well as by slightly defocusing the laser. These results should help optimize cell tagging for genotype-phenotype mapping in optical pooled screens, as well as for other applications.