DNA microscopy Optics-free spatio-genetic imaging by a stand-alone chemical reaction, bioRxiv, 2018-11-19
AbstractAnalyzing the spatial organization of molecules in cells and tissues is a cornerstone of biological research and clinical practice. However, despite enormous progress in profiling the molecular constituents of cells, spatially mapping these constituents remains a disjointed and machinery-intensive process, relying on either light microscopy or direct physical registration and capture. Here, we demonstrate DNA microscopy, a new imaging modality for scalable, optics-free mapping of relative biomolecule positions. In DNA microscopy of transcripts, transcript molecules are tagged in situ with randomized nucleotides, labeling each molecule uniquely. A second in situ reaction then amplifies the tagged molecules, concatenates the resulting copies, and adds new randomized nucleotides to uniquely label each concatenation event. An algorithm decodes molecular proximities from these concatenated sequences, and infers physical images of the original transcripts at cellular resolution. Because its imaging power derives entirely from diffusive molecular dynamics, DNA microscopy constitutes a chemically encoded microscopy system.
biorxiv bioengineering 200-500-users 2018Universal Light-Sheet Generation with Field Synthesis, bioRxiv, 2018-09-26
AbstractWe introduce Field Synthesis, a theorem that can be used to synthesize any scanned or dithered light-sheet, including those used in lattice light-sheet microscopy (LLSM), from an incoherent superposition of one-dimensional intensity distributions. This user-friendly and modular approach offers a drastically simplified optical design, higher light-throughput, simultaneous multicolor illumination, and a 100% spatial duty cycle, thereby providing uncompromised biological imaging with decreased rates of photobleaching.
biorxiv bioengineering 100-200-users 2018MAPLE a Modular Automated Platform for Large-scale Experiments, a low-cost robot for integrated animal-handling and phenotyping, bioRxiv, 2017-12-25
AbstractGenetic model system animals have significant scientific value in part because of large-scale experiments like screens, but performing such experiments over long time periods by hand is arduous and risks errors. Thus the field is poised to benefit from automation, just as molecular biology did from liquid-handling robots. We developed a Modular Automated Platform for Large-scale Experiments (MAPLE), a Drosophila-handling robot capable of conducting lab tasks and experiments. We demonstrate MAPLE’s ability to accelerate the collection of virgin female flies (a pervasive experimental chore in fly genetics) and assist high-throughput phenotyping assays. Using MAPLE to autonomously run a novel social interaction experiment, we found that 1) pairs of flies exhibit persistent idiosyncrasies in affiliative behavior, 2) these dyad-specific interactions require olfactory and visual cues, and 3) social interaction network structure is topologically stable over time. These diverse examples demonstrate MAPLE’s versatility as a general platform for conducting fly science automatically.
biorxiv bioengineering 0-100-users 2017Exponential fluorescent amplification of individual RNAs using clampFISH probes, bioRxiv, 2017-12-06
AbstractNon-enzymatic, high-gain signal amplification methods with single-cell, single-molecule resolution are in great need. We present click-amplifying FISH (clampFISH) for the fluorescent detection of RNA that combines the specificity of oligonucleotides with bioorthogonal click chemistry in order to achieve high specificity and extremely high-gain (>400x) signal amplification. We show that clampFISH signal enables detection with low magnification microscopy and separation of cells by RNA levels via flow cytometry. Additionally, we show that the modular design of clampFISH probes enables multiplexing, that the locking mechanism prevents probe detachment in expansion microscopy, and that clampFISH works in tissue samples.
biorxiv bioengineering 200-500-users 2017Directed evolution of TurboID for efficient proximity labeling in living cells and organisms, bioRxiv, 2017-10-03
AbstractProtein interaction networks and protein compartmentation underlie every signaling process and regulatory mechanism in cells. Recently, proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, the two enzymes commonly used for PL come with tradeoffs – BioID is slow, requiring tagging times of 18-24 hours, while APEX peroxidase uses substrates that have limited cell permeability and high toxicity. To address these problems, we used yeast display-based directed evolution to engineer two mutants of biotin ligase, TurboID and miniTurbo, with much greater catalytic efficiency than BioID, and the ability to carry out PL in cells in much shorter time windows (as little as 10 minutes) with non-toxic and easily deliverable biotin. In addition to shortening PL time by 100-fold and increasing PL yield in cell culture, TurboID enabled biotin-based PL in new settings, including yeast, Drosophila, and C. elegans.
biorxiv bioengineering 0-100-users 2017Corrigendum and follow-up Whole genome sequencing of multiple CRISPR-edited mouse lines suggests no excess mutations, bioRxiv, 2017-06-24
Our previous publication suggested CRISPR-Cas9 editing at the zygotic stage might unexpectedly introduce a multitude of subtle but unintended mutations, an interpretation that not surprisingly raised numerous questions. The key issue is that since parental lines were not available, might the reported variants have been inherited? To expand upon the limited available whole genome data on whether CRISPR-edited mice show more genetic variation, whole-genome sequencing was performed on two other mouse lines that had undergone a CRISPR-editing procedure. Again, parents were not available for either the Capn5 nor Fblim1 CRISPR-edited mouse lines, so strain controls were examined. Additionally, we also include verification of variants detected in the initial mouse line. Taken together, these whole-genome-sequencing-level results support the idea that in specific cases, CRISPR-Cas9 editing can precisely edit the genome at the organismal level and may not introduce numerous, unintended, off-target mutations.
biorxiv bioengineering 200-500-users 2017