Mapping the genomic landscape of CRISPR-Cas9 cleavage.

RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.

The evolution of a series of behavioral traits is associated with autism-risk genes in cavefish.

BACKGROUND: An essential question in evolutionary biology is whether shifts in a set of polygenic behaviors share a genetic basis across species. Such a behavioral shift is seen in the cave-dwelling Mexican tetra, Astyanax mexicanus. Relative to surface-dwelling conspecifics, cavefish do not school (asocial), are hyperactive and sleepless, adhere to a particular vibration stimulus (imbalanced attention), behave repetitively, and show elevated stress hormone levels. Interestingly, these traits largely overlap with the core symptoms of human autism spectrum disorder (ASD), raising the possibility that these behavioral traits are underpinned by a similar set of genes (i.e. a repeatedly used suite of genes). RESULT: Here, we explored whether modification of ASD-risk genes underlies cavefish evolution. Transcriptomic analyses revealed that > 58.5% of 3152 cavefish orthologs to ASD-risk genes are significantly up- or down-regulated in the same direction as genes in postmortem brains from ASD patients. Enrichment tests suggest that ASD-risk gene orthologs in A. mexicanus have experienced more positive selection than other genes across the genome. Notably, these positively selected cavefish ASD-risk genes are enriched for pathways involved in gut function, inflammatory diseases, and lipid/energy metabolism, similar to symptoms that frequently coexist in ASD patients. Lastly, ASD drugs mitigated cavefish's ASD-like behaviors, implying shared aspects of neural processing. CONCLUSION: Overall, our study indicates that ASD-risk genes and associated pathways (especially digestive, immune and metabolic pathways) may be repeatedly used for shifts in polygenic behaviors across evolutionary time.

SHAPE-Seq 2.0: systematic optimization and extension of high-throughput chemical probing of RNA secondary structure with next generation sequencing.

RNA structure is a primary determinant of its function, and methods that merge chemical probing with next generation sequencing have created breakthroughs in the throughput and scale of RNA structure characterization. However, little work has been done to examine the effects of library preparation and sequencing on the measured chemical probe reactivities that encode RNA structural information. Here, we present the first analysis and optimization of these effects for selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). We first optimize SHAPE-Seq, and show that it provides highly reproducible reactivity data over a wide range of RNA structural contexts with no apparent biases. As part of this optimization, we present SHAPE-Seq v2.0, a 'universal' method that can obtain reactivity information for every nucleotide of an RNA without having to use or introduce a specific reverse transcriptase priming site within the RNA. We show that SHAPE-Seq v2.0 is highly reproducible, with reactivity data that can be used as constraints in RNA folding algorithms to predict structures on par with those generated using data from other SHAPE methods. We anticipate SHAPE-Seq v2.0 to be broadly applicable to understanding the RNA sequence-structure relationship at the heart of some of life's most fundamental processes.

ß2 integrins impose a mechanical checkpoint on macrophage phagocytosis.

Phagocytosis is an intensely physical process that depends on the mechanical properties of both the phagocytic cell and its chosen target. Here, we employed differentially deformable hydrogel microparticles to examine the role of cargo rigidity in the regulation of phagocytosis by macrophages. Whereas stiff cargos elicited canonical phagocytic cup formation and rapid engulfment, soft cargos induced an architecturally distinct response, characterized by filamentous actin protrusions at the center of the contact site, slower cup advancement, and frequent phagocytic stalling. Using phosphoproteomics, we identified ß2 integrins as critical mediators of this mechanically regulated phagocytic switch. Macrophages lacking ß2 integrins or their downstream effectors, Talin1 and Vinculin, exhibited specific defects in phagocytic cup architecture and selective suppression of stiff cargo uptake. We conclude that integrin signaling serves as a mechanical checkpoint during phagocytosis to pair cargo rigidity to the appropriate mode of engulfment.

Single-cell topographical profiling of the immune synapse reveals a biomechanical signature of cytotoxicity.

Immune cells have intensely physical lifestyles characterized by structural plasticity and force exertion. To investigate whether specific immune functions require stereotyped mechanical outputs, we used super-resolution traction force microscopy to compare the immune synapses formed by cytotoxic T cells with contacts formed by other T cell subsets and by macrophages. T cell synapses were globally compressive, which was fundamentally different from the pulling and pinching associated with macrophage phagocytosis. Spectral decomposition of force exertion patterns from each cell type linked cytotoxicity to compressive strength, local protrusiveness, and the induction of complex, asymmetric topography. These features were validated as cytotoxic drivers by genetic disruption of cytoskeletal regulators, live imaging of synaptic secretion, and in silico analysis of interfacial distortion. Synapse architecture and force exertion were sensitive to target stiffness and size, suggesting that the mechanical potentiation of killing is biophysically adaptive. We conclude that cellular cytotoxicity and, by implication, other effector responses are supported by specialized patterns of efferent force.

Plasma membrane abundance dictates phagocytic capacity and functional cross-talk in myeloid cells.

Professional phagocytes like neutrophils and macrophages tightly control what they consume, how much they consume, and when they move after cargo uptake. We show that plasma membrane abundance is a key arbiter of these cellular behaviors. Neutrophils and macrophages lacking the G protein subunit Gß4 exhibited profound plasma membrane expansion, accompanied by marked reduction in plasma membrane tension. These biophysical changes promoted the phagocytosis of bacteria, fungus, apoptotic corpses, and cancer cells. We also found that Gß4-deficient neutrophils are defective in the normal inhibition of migration following cargo uptake. Sphingolipid synthesis played a central role in these phenotypes by driving plasma membrane accumulation in cells lacking Gß4. In Gß4 knockout mice, neutrophils not only exhibited enhanced phagocytosis of inhaled fungal conidia in the lung but also increased trafficking of engulfed pathogens to other organs. Together, these results reveal an unexpected, biophysical control mechanism central to myeloid functional decision-making.

Intravenous Iron for Heart Failure: Updated Systematic Review and Meta-Analysis.

While the administration of intravenous (IV) iron to those with heart failure has been implicated to be associated with a possible reduction in hospitalizations and improvement in symptoms, a recent large multicenter trial only showed modest benefits in reducing hospitalization, necessitating the updated systematic review. We conducted a systematic review and meta-analysis, searching the MEDLINE and EMBASE database until January 9, 2023. Outcomes included total heart failure hospitalizations, first heart failure hospitalization, six-minute walk test (6MWT) distance, and incidence of infection. There were 13 studies with 3410 participants (1,790 with IV iron). Pooled analysis that reported the incidence of cardiovascular death showed that patients with IV iron did not have significantly lower odds of cardiovascular death or first heart failure hospitalization. In contrast, those who received IV iron had significantly lower total heart failure hospitalization (pooled odds ratio (OR) 0.63, 95% confidence interval (CI) 0.44-0.90, I2 59.0%, P = .017) and a composite of cardiovascular death and first heart failure hospitalization (pooled OR 0.55, 95% CI 0.47-0.64, I2 0%, P = .656). While the efficacy is modest, IV iron therapy could be associated with reduced hospitalization for heart failure without significant adverse events.

Topographical analysis of immune cell interactions reveals a biomechanical signature for immune cytolysis.

Immune cells live intensely physical lifestyles characterized by structural plasticity, mechanosensitivity, and force exertion. Whether specific immune functions require stereotyped patterns of mechanical output, however, is largely unknown. To address this question, we used super-resolution traction force microscopy to compare cytotoxic T cell immune synapses with contacts formed by other T cell subsets and macrophages. T cell synapses were globally and locally protrusive, which was fundamentally different from the coupled pinching and pulling of macrophage phagocytosis. By spectrally decomposing the force exertion patterns of each cell type, we associated cytotoxicity with compressive strength, local protrusiveness, and the induction of complex, asymmetric interfacial topographies. These features were further validated as cytotoxic drivers by genetic disruption of cytoskeletal regulators, direct imaging of synaptic secretory events, and in silico analysis of interfacial distortion. We conclude that T cell-mediated killing and, by implication, other effector responses are supported by specialized patterns of efferent force.

Plasma membrane abundance dictates phagocytic capacity and functional crosstalk in myeloid cells.

Professional phagocytes like neutrophils and macrophages tightly control what they eat, how much they eat, and when they move after eating. We show that plasma membrane abundance is a key arbiter of these cellular behaviors. Neutrophils and macrophages lacking the G-protein subunit Gb4 exhibit profound plasma membrane expansion due to enhanced production of sphingolipids. This increased membrane allocation dramatically enhances phagocytosis of bacteria, fungus, apoptotic corpses, and cancer cells. Gb4 deficient neutrophils are also defective in the normal inhibition of migration following cargo uptake. In Gb4 knockout mice, myeloid cells exhibit enhanced phagocytosis of inhaled fungal conidia in the lung but also increased trafficking of engulfed pathogens to other organs. These results reveal an unexpected, biophysical control mechanism lying at the heart of myeloid functional decision-making.

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish.

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among these traits, foraging behavior is one of the useful windows into functional advantages of behavioral trait evolution. Presented herein are updated methods for analyzing vibration attraction behavior (VAB: an adaptive foraging behavior) and imaging of associated mechanosensors of cave-adapted tetra, Astyanax mexicanus. In addition, methods are presented for high-throughput tracking of a series of additional cavefish behaviors including hyperactivity and sleep-loss. Cavefish also show asociality, repetitive behavior and higher anxiety. Therefore, cavefish serve as an animal model for evolved behaviors. These methods use free-software and custom-made scripts that can be applied to other types of behavior. These methods provide practical and cost-effective alternatives to commercially available tracking software.

NmeCas9 is an intrinsically high-fidelity genome-editing platform.

BACKGROUND: The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery. RESULTS: Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5'-N4GATT-3'), for NmeCas9 genome editing in human cells. CONCLUSIONS: Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.