Exonuclease-enhanced prime editors.

Prime editing (PE) is a powerful gene-editing technique based on targeted gRNA-templated reverse transcription and integration of the de novo synthesized single-stranded DNA. To circumvent one of the main bottlenecks of the method, the competition of the reverse-transcribed 3' flap with the original 5' flap DNA, we generated an enhanced fluorescence-activated cell sorting reporter cell line to develop an exonuclease-enhanced PE strategy ('Exo-PE') composed of an improved PE complex and an aptamer-recruited DNA-exonuclease to remove the 5' original DNA flap. Exo-PE achieved better overall editing efficacy than the reference PE2 strategy for insertions ≥30 base pairs in several endogenous loci and cell lines while maintaining the high editing precision of PE2. By enabling the precise incorporation of larger insertions, Exo-PE complements the growing palette of different PE tools and spurs additional refinements of the PE machinery.

A reversible state of hypometabolism in a human cellular model of sporadic Parkinson's disease.

Sporadic Parkinson's Disease (sPD) is a progressive neurodegenerative disorder caused by multiple genetic and environmental factors. Mitochondrial dysfunction is one contributing factor, but its role at different stages of disease progression is not fully understood. Here, we showed that neural precursor cells and dopaminergic neurons derived from induced pluripotent stem cells (hiPSCs) from sPD patients exhibited a hypometabolism. Further analysis based on transcriptomics, proteomics, and metabolomics identified the citric acid cycle, specifically the α-ketoglutarate dehydrogenase complex (OGDHC), as bottleneck in sPD metabolism. A follow-up study of the patients approximately 10 years after initial biopsy demonstrated a correlation between OGDHC activity in our cellular model and the disease progression. In addition, the alterations in cellular metabolism observed in our cellular model were restored by interfering with the enhanced SHH signal transduction in sPD. Thus, inhibiting overactive SHH signaling may have potential as neuroprotective therapy during early stages of sPD.

Genetically encoded barcodes for correlative volume electron microscopy.

While genetically encoded reporters are common for fluorescence microscopy, equivalent multiplexable gene reporters for electron microscopy (EM) are still scarce. Here, by installing a variable number of fixation-stable metal-interacting moieties in the lumen of encapsulin nanocompartments of different sizes, we developed a suite of spherically symmetric and concentric barcodes (EMcapsulins) that are readable by standard EM techniques. Six classes of EMcapsulins could be automatically segmented and differentiated. The coding capacity was further increased by arranging several EMcapsulins into distinct patterns via a set of rigid spacers of variable length. Fluorescent EMcapsulins were expressed to monitor subcellular structures in light and EM. Neuronal expression in Drosophila and mouse brains enabled the automatic identification of genetically defined cells in EM. EMcapsulins are compatible with transmission EM, scanning EM and focused ion beam scanning EM. The expandable palette of genetically controlled EM-readable barcodes can augment anatomical EM images with multiplexed gene expression maps.

Intron-encoded cistronic transcripts for minimally invasive monitoring of coding and non-coding RNAs.

Despite their fundamental role in assessing (patho)physiological cell states, conventional gene reporters can follow gene expression but leave scars on the proteins or substantially alter the mature messenger RNA. Multi-time-point measurements of non-coding RNAs are currently impossible without modifying their nucleotide sequence, which can alter their native function, half-life and localization. Thus, we developed the intron-encoded scarless programmable extranuclear cistronic transcript (INSPECT) as a minimally invasive transcriptional reporter embedded within an intron of a gene of interest. Post-transcriptional excision of INSPECT results in the mature endogenous RNA without sequence alterations and an additional engineered transcript that leaves the nucleus by hijacking the nuclear export machinery for subsequent translation into a reporter or effector protein. We showcase its use in monitoring interleukin-2 (IL2) after T cell activation and tracking the transcriptional dynamics of the long non-coding RNA (lncRNA) NEAT1 during CRISPR interference-mediated perturbation. INSPECT is a method for monitoring gene transcription without altering the mature lncRNA or messenger RNA of the target of interest.

Photoacoustic Neuroimaging - Perspectives on a Maturing Imaging Technique and its Applications in Neuroscience.

A prominent goal of neuroscience is to improve our understanding of how brain structure and activity interact to produce perception, emotion, behavior, and cognition. The brain's network activity is inherently organized in distinct spatiotemporal patterns that span scales from nanometer-sized synapses to meter-long nerve fibers and millisecond intervals between electrical signals to decades of memory storage. There is currently no single imaging method that alone can provide all the relevant information, but intelligent combinations of complementary techniques can be effective. Here, we thus present the latest advances in biomedical and biological engineering on photoacoustic neuroimaging in the context of complementary imaging techniques. A particular focus is placed on recent advances in whole-brain photoacoustic imaging in rodent models and its influential role in bridging the gap between fluorescence microscopy and more non-invasive techniques such as magnetic resonance imaging (MRI). We consider current strategies to address persistent challenges, particularly in developing molecular contrast agents, and conclude with an overview of potential future directions for photoacoustic neuroimaging to provide deeper insights into healthy and pathological brain processes.

Non-invasive and high-throughput interrogation of exon-specific isoform expression.

Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

Noninvasive visualization of electrical conductivity in tissues at the micrometer scale.

Despite its importance in regulating cellular or tissue function, electrical conductivity can only be visualized in tissue indirectly as voltage potentials using fluorescent techniques, or directly with radio waves. These either requires invasive procedures like genetic modification or suffers from limited resolution. Here, we introduce radio-frequency thermoacoustic mesoscopy (RThAM) for the noninvasive imaging of conductivity by exploiting the direct absorption of near-field ultrashort radio-frequency pulses to stimulate the emission of broadband ultrasound waves. Detection of ultrasound rather than radio waves enables micrometer-scale resolutions, over several millimeters of tissue depth. We confirm an imaging resolution of <30 µm in phantoms and demonstrate microscopic imaging of conductivity correlating to physical structures in 1- and 512-cell zebrafish embryos, as well as larvae. These results support RThAM as a promising method for high-resolution, label-free assessment of conductivity in tissues.

Genetically encodable materials for non-invasive biological imaging.

Many questions in basic biology and medicine require the ability to visualize the function of specific cells and molecules inside living organisms. In this context, technologies such as ultrasound, optoacoustics and magnetic resonance provide non-invasive imaging access to deep-tissue regions, as used in many laboratories and clinics to visualize anatomy and physiology. In addition, recent work has enabled these technologies to image the location and function of specific cells and molecules inside the body by coupling the physics of sound waves, nuclear spins and light absorption to unique protein-based materials. These materials, which include air-filled gas vesicles, capsid-like nanocompartments, pigment-producing enzymes and transmembrane transporters, enable new forms of biomolecular and cellular contrast. The ability of these protein-based contrast agents to be genetically encoded and produced by cells creates opportunities for unprecedented in vivo studies of cellular function, while their amenability to genetic engineering enables atomic-level design of their physical, chemical and biological properties.

Nucleus size and DNA accessibility are linked to the regulation of paraspeckle formation in cellular differentiation.

BACKGROUND: Many long noncoding RNAs (lncRNAs) have been implicated in general and cell type-specific molecular regulation. Here, we asked what underlies the fundamental basis for the seemingly random appearance of nuclear lncRNA condensates in cells, and we sought compounds that can promote the disintegration of lncRNA condensates in vivo. RESULTS: As a basis for comparing lncRNAs and cellular properties among different cell types, we screened lncRNAs in human pluripotent stem cells (hPSCs) that were differentiated to an atlas of cell lineages. We found that paraspeckles, which form by aggregation of the lncRNA NEAT1, are scaled by the size of the nucleus, and that small DNA-binding molecules promote the disintegration of paraspeckles and other lncRNA condensates. Furthermore, we found that paraspeckles regulate the differentiation of hPSCs. CONCLUSIONS: Positive correlation between the size of the nucleus and the number of paraspeckles exist in numerous types of human cells. The tethering and structure of paraspeckles, as well as other lncRNAs, to the genome can be disrupted by small molecules that intercalate in DNA. The structure-function relationship of lncRNAs that regulates stem cell differentiation is likely to be determined by the dynamics of nucleus size and binding site accessibility.

Calcium neuroimaging in behaving zebrafish larvae using a turn-key light field camera.

Reconstructing a three-dimensional scene from multiple simultaneously acquired perspectives (the light field) is an elegant scanless imaging concept that can exceed the temporal resolution of currently available scanning-based imaging methods for capturing fast cellular processes. We tested the performance of commercially available light field cameras on a fluorescent microscopy setup for monitoring calcium activity in the brain of awake and behaving reporter zebrafish larvae. The plenoptic imaging system could volumetrically resolve diverse neuronal response profiles throughout the zebrafish brain upon stimulation with an aversive odorant. Behavioral responses of the reporter fish could be captured simultaneously together with depth-resolved neuronal activity. Overall, our assessment showed that with some optimizations for fluorescence microscopy applications, commercial light field cameras have the potential of becoming an attractive alternative to custom-built systems to accelerate molecular imaging research on cellular dynamics.