PAM-flexible genome editing with an engineered chimeric Cas9.

CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.

PAM-Flexible Genome Editing with an Engineered Chimeric Cas9.

CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN PAM preference, with the N-terminus of Sc++, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse NNN PAMs and disease-related loci for potential therapeutic applications. In total, the unique approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.

Recent Microscopy Advances and the Applications to Huntington's Disease Research.

Huntingtin is a 3144 amino acid protein defined as a scaffold protein with many intracellular locations that suggest functions in these compartments. Expansion of the CAG DNA tract in the huntingtin first exon is the cause of Huntington's disease. An important tool in understanding the biological functions of huntingtin is molecular imaging at the single-cell level by microscopy and nanoscopy. The evolution of these technologies has accelerated since the Nobel Prize in Chemistry was awarded in 2014 for super-resolution nanoscopy. We are in a new era of light imaging at the single-cell level, not just for protein location, but also for protein conformation and biochemical function. Large-scale microscopy-based screening is also being accelerated by a coincident development of machine-based learning that offers a framework for truly unbiased data acquisition and analysis at very large scales. This review will summarize the newest technologies in light, electron, and atomic force microscopy in the context of unique challenges with huntingtin cell biology and biochemistry.

Mod3D: A low-cost, flexible modular system of live-cell microscopy chambers and holders.

Live-cell microscopy imaging typically involves the use of high-quality glass-bottom chambers that allow cell culture, gaseous buffer exchange and optical properties suitable for microscopy applications. However, commercial sources of these chambers can add significant annual costs to cell biology laboratories. Consumer products in three-dimensional printing technology, for both Filament Deposition Modeling (FDM) and Masked Stereo Lithography (MSLA), have resulted in more biomedical research labs adopting the use of these devices for prototyping and manufacturing of lab plastic-based items, but rarely consumables. Here we describe a modular, live-cell chamber with multiple design options that can be mixed per experiment. Single reusable carriers and the use of biodegradable plastics, in a hybrid of FDM and MSLA manufacturing methods, reduce plastic waste. The system is easy to adapt to bespoke designs, with concept-to-prototype in a single day, offers significant cost savings to the users over commercial sources, and no loss in dimensional quality or reliability.

The impact of the COVID-19 pandemic on perceived publication pressure among academic researchers in Canada.

The phenomenon of "publish-or-perish" in academia, spurred on by limited funding and academic positions, has led to increased competition and pressure on academics to publish. Publication pressure has been linked with multiple negative outcomes, including increased academic misconduct and researcher burnout. COVID-19 has disrupted research worldwide, leading to lost research time and increased anxiety amongst researchers. The objective of this study was to examine how COVID-19 has impacted perceived publication pressure amongst academic researchers in Canada. We used the revised Publication Pressure Questionnaire, in addition to Likert-type questions to discern respondents' beliefs and concerns about the impact of COVID-19 on academic publishing. We found that publication pressure increased across academic researchers in Canada following the pandemic, with respondents reporting increased stress, increased pessimism, and decreased access to support related to publishing. Doctoral students reported the highest levels of stress and pessimism, while principal investigators had the most access to publication support. There were no significant differences in publication pressure reported between different research disciplines. Women and non-binary or genderfluid respondents reported higher stress and pessimism than men. We also identified differences in perceived publication pressure based on respondents' publication frequency and other demographic factors, including disability and citizenship status. Overall, we document a snapshot of perceived publication pressure in Canada across researchers of different academic career stages and disciplines. This information can be used to guide the creation of researcher supports, as well as identify groups of researchers who may benefit from targeted resources.

Functional characterization of variants of unknown significance in a spinocerebellar ataxia patient using an unsupervised machine learning pipeline.

CAG-expanded ATXN7 has been previously defined in the pathogenesis of spinocerebellar ataxia type 7 (SCA7), a polyglutamine expansion autosomal dominant cerebellar ataxia. Pathology in SCA7 occurs as a result of a CAG triplet repeat expansion in excess of 37 in the first exon of ATXN7, which encodes ataxin-7. SCA7 presents clinically with spinocerebellar ataxia and cone-rod dystrophy. Here, we present a novel spinocerebellar ataxia variant occurring in a patient with mutations in both ATXN7 and TOP1MT, which encodes mitochondrial topoisomerase I (top1mt). Using machine-guided, unbiased microscopy image analysis, we demonstrate alterations in ataxin-7 subcellular localization, and through high-fidelity measurements of cellular respiration, bioenergetic defects in association with top1mt mutations. We identify ataxin-7 Q35P and top1mt R111W as deleterious mutations, potentially contributing to disease states. We recapitulate our mutations through Drosophila genetic models. Our work provides important insight into the cellular biology of ataxin-7 and top1mt and offers insight into the pathogenesis of spinocerebellar ataxia applicable to multiple subtypes of the illness. Moreover, our study demonstrates an effective pipeline for the characterization of previously unreported genetic variants at the level of cell biology.

Huntingtin structure is orchestrated by HAP40 and shows a polyglutamine expansion-specific interaction with exon 1.

Huntington's disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo and in cell models that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 Å cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The exon 1 region of HTT is dynamic but shows greater conformational variety in the polyglutamine expanded mutant than wildtype exon 1. Our data provide a foundation for future functional and drug discovery studies targeting Huntington's disease and illuminate the structural consequences of HTT polyglutamine expansion.

Spinocerebellar Ataxia Type 1 protein Ataxin-1 is signaled to DNA damage by ataxia-telangiectasia mutated kinase.

Spinocerebellar Ataxia Type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion in the ataxin-1 protein. Recent genetic correlational studies have implicated DNA damage repair pathways in modifying the age at onset of disease symptoms in SCA1 and Huntington's Disease, another polyglutamine expansion disease. We demonstrate that both endogenous and transfected ataxin-1 localizes to sites of DNA damage, which is impaired by polyglutamine expansion. This response is dependent on ataxia-telangiectasia mutated (ATM) kinase activity. Further, we characterize an ATM phosphorylation motif within ataxin-1 at serine 188. We show reduction of the Drosophila ATM homolog levels in a ATXN1[82Q] Drosophila model through shRNA or genetic cross ameliorates motor symptoms. These findings offer a possible explanation as to why DNA repair was implicated in SCA1 pathogenesis by past studies. The similarities between the ataxin-1 and the huntingtin responses to DNA damage provide further support for a shared pathogenic mechanism for polyglutamine expansion diseases.

DNA Repair in Huntington's Disease and Spinocerebellar Ataxias: Somatic Instability and Alternative Hypotheses.

The use of genome wide association studies (GWAS) in Huntington's disease (HD) research, driven by unbiased human data analysis, has transformed the focus of new targets that could affect age at onset. While there is a significant depth of information on DNA damage repair, with many drugs and drug targets, most of this development has taken place in the context of cancer therapy. DNA damage repair in neurons does not rely on DNA replication correction mechanisms. However, there is a strong connection between DNA repair and neuronal metabolism, mediated by nucleotide salvaging and the poly ADP-ribose (PAR) response, and this connection has been implicated in other age-onset neurodegenerative diseases. Validation of leads including the mismatch repair protein MSH3, and interstrand cross-link repair protein FAN1, suggest the mechanism is driven by somatic CAG instability, which is supported by the protective effect of CAA substitutions in the CAG tract. We currently do not understand: how somatic instability is triggered; the state of DNA damage within expanding alleles in the brain; whether this damage induces mismatch repair and interstrand cross-link pathways; whether instability mediates toxicity, and how this relates to human ageing. We discuss DNA damage pathways uncovered by HD GWAS, known roles of other polyglutamine disease proteins in DNA damage repair, and a panel of hypotheses for pathogenic mechanisms.

Shedding a new light on Huntington's disease: how blood can both propagate and ameliorate disease pathology.

Huntington's disease (HD) is a monogenic neurodegenerative disorder resulting from a mutation in the huntingtin gene. This leads to the expression of the mutant huntingtin protein (mHTT) which provokes pathological changes in both the central nervous system (CNS) and periphery. Accumulating evidence suggests that mHTT can spread between cells of the CNS but here, we explored the possibility that mHTT could also propagate and cause pathology via the bloodstream. For this, we used a parabiosis approach to join the circulatory systems of wild-type (WT) and zQ175 mice. After surgery, we observed mHTT in the plasma and circulating blood cells of WT mice and post-mortem analyses revealed the presence of mHTT aggregates in several organs including the liver, kidney, muscle and brain. The presence of mHTT in the brain was accompanied by vascular abnormalities, such as a reduction of Collagen IV signal intensity and altered vessel diameter in the striatum, and changes in expression of Glutamic acid decarboxylase 65/67 (GAD65-67) in the cortex. Conversely, we measured reduced pathology in zQ175 mice by decreased mitochondrial impairments in peripheral organs, restored vessel diameter in the cortex and improved expression of Dopamine- and cAMP-regulated phosphoprotein 32 (DARPP32) in striatal neurons. Collectively, these results demonstrate that circulating mHTT can disseminate disease, but importantly, that healthy blood can dilute pathology. These findings have significant implications for the development of therapies in HD.

Development of a knowledge translation platform for ataxia: Impact on readers and volunteer contributors.

BACKGROUND: Dissemination of accurate health research information to patients and families has become increasingly important with the rise of the internet as a means of finding health information. However, the public faces several barriers to accessing research information, including paywalls and technical jargon. One method to bridge this gap between patients, families, and research is using lay summaries. SCAsource is an online knowledge translation platform where peer-reviewed research papers on ataxia are translated into lay summaries. This online platform was launched in September 2018, with the goal of making ataxia research more accessible and understandable to patients and families. A secondary goal is to provide opportunities for ataxia researchers to develop and hone their knowledge translation skills, altogether improving the quality of patient communication in the ataxia community. AIM: The aim of this study was to measure the impact of SCAsource on its readers and volunteer contributors after one year of activity. This is to ensure SCAsource is meeting its goals of (1) improving access and understanding of ataxia research to lay audiences, and (2) improving knowledge translation skills of volunteer contributors. METHODS: Two online surveys were launched, one for readers and one for volunteers. Each survey had a combination of multiple-choice, Likert-scale type, and open-ended short-answer questions. Descriptive quantitative analysis was used for respondent characteristics and Likert-type data. A grounded theory coding approach was used to analyze narrative feedback data. RESULTS: We found that SCAsource has mutually beneficial outcomes for both lay person readers and volunteer contributors. Readers have an increased understanding of ataxia research and access to up-to-date information on recent publications. Volunteers develop knowledge translation skills and have increased confidence in communicating results to lay audiences. Areas of improvement were identified to be incorporated into the platform. CONCLUSION: We demonstrated that SCAsource improves access to information and understanding of research to lay audiences, while providing opportunities for researchers to develop knowledge translation skills. This framework can potentially be used by other rare disease organizations to launch and evaluate their own knowledge translation websites.

Single Cell Technologies Define New Therapeutic Avenues for Huntington's Disease.

Using two independent single-cell transcriptomics technologies, Lee et al. have cataloged transcriptional changes in the subset of striatal neurons hit hardest in Huntington's disease. One downregulated pathway, oxidative phosphorylation, may also explain their observed release of mitochondrial-encoded RNAs.

DNA Repair Signaling of Huntingtin: The Next Link Between Late-Onset Neurodegenerative Disease and Oxidative DNA Damage.

A new hypothesis for the mechanism of Huntington's disease (HD) is driven by a small molecule lead that may connect age-associated reactive oxygen stress, oxidative DNA damage, and mitochondrial dysfunction. These pathways have also recently been defined in genome-wide association studies of cytosine-adenine-guanine-expansion polyglutamine neurodegenerative diseases, including HD and the spinocerebellar ataxias. We discuss how N6-furfuryladenine (N6FFA) nucleotide salvage and role as a kinase neosubstrate may have important mechanistic implications for both HD and familial Parkinson's disease. N6FFA highlights a mechanism of how energy dysregulation and protein misfolding in neurodegeneration may be the effect of age-associated reactive oxygen species damage to DNA and part of a feedback loop augmenting with aging.

High-mobility group box 1 links sensing of reactive oxygen species by huntingtin to its nuclear entry.

Huntington's disease (HD) is a neurodegenerative, age-onset disorder caused by a CAG DNA expansion in exon 1 of the HTT gene, resulting in a polyglutamine expansion in the huntingtin protein. Nuclear accumulation of mutant huntingtin is a hallmark of HD, resulting in elevated mutant huntingtin levels in cell nuclei. Huntingtin is normally retained at the endoplasmic reticulum via its N17 amphipathic α-helix domain but is released by oxidation of Met-8 during reactive oxygen species (ROS) stress. Huntingtin enters the nucleus via an importin ß1- and 2-dependent proline-tyrosine nuclear localization signal (PY-NLS), which has a unique intervening sequence in huntingtin. Here, we have identified the high-mobility group box 1 (HMGB1) protein as an interactor of the intervening sequence within the PY-NLS. Nuclear levels of HMGB1 positively correlated with varying levels of nuclear huntingtin in both HD and normal human fibroblasts. We also found that HMGB1 interacts with the huntingtin N17 region and that this interaction is enhanced by the presence of ROS and phosphorylation of critical serine residues in the N17 region. We conclude that HMGB1 is a huntingtin N17/PY-NLS ROS-dependent interactor, and this protein bridging is essential for relaying ROS sensing by huntingtin to its nuclear entry during ROS stress. ROS may therefore be a critical age-onset stress that triggers nuclear accumulation of mutant huntington in Huntington's disease.

A patient-derived cellular model for Huntington's disease reveals phenotypes at clinically relevant CAG lengths.

The huntingtin protein participates in several cellular processes that are disrupted when the polyglutamine tract is expanded beyond a threshold of 37 CAG DNA repeats in Huntington's disease (HD). Cellular biology approaches to understand these functional disruptions in HD have primarily focused on cell lines with synthetically long CAG length alleles that clinically represent outliers in this disease and a more severe form of HD that lacks age onset. Patient-derived fibroblasts are limited to a finite number of passages before succumbing to cellular senescence. We used human telomerase reverse transcriptase (hTERT) to immortalize fibroblasts taken from individuals of varying age, sex, disease onset, and CAG repeat length, which we have termed TruHD cells. TruHD cells display classic HD phenotypes of altered morphology, size and growth rate, increased sensitivity to oxidative stress, aberrant adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratios, and hypophosphorylated huntingtin protein. We additionally observed dysregulated reactive oxygen species (ROS)-dependent huntingtin localization to nuclear speckles in HD cells. We report the generation and characterization of a human, clinically relevant cellular model for investigating disease mechanisms in HD at the single-cell level, which, unlike transformed cell lines, maintains functions critical for huntingtin transcriptional regulation and genomic integrity.

N6-Furfuryladenine is protective in Huntington's disease models by signaling huntingtin phosphorylation.

The huntingtin N17 domain is a modulator of mutant huntingtin toxicity and is hypophosphorylated in Huntington's disease (HD). We conducted high-content analysis to find compounds that could restore N17 phosphorylation. One lead compound from this screen was N6-furfuryladenine (N6FFA). N6FFA was protective in HD model neurons, and N6FFA treatment of an HD mouse model corrects HD phenotypes and eliminates cortical mutant huntingtin inclusions. We show that N6FFA restores N17 phosphorylation levels by being salvaged to a triphosphate form by adenine phosphoribosyltransferase (APRT) and used as a phosphate donor by casein kinase 2 (CK2). N6FFA is a naturally occurring product of oxidative DNA damage. Phosphorylated huntingtin functionally redistributes and colocalizes with CK2, APRT, and N6FFA DNA adducts at sites of induced DNA damage. We present a model in which this natural product compound is salvaged to provide a triphosphate substrate to signal huntingtin phosphorylation via CK2 during low-ATP stress under conditions of DNA damage, with protective effects in HD model systems.

Atraumatic versus conventional lumbar puncture needles: a systematic review and meta-analysis.

BACKGROUND: Atraumatic needles have been proposed to lower complication rates after lumbar puncture. However, several surveys indicate that clinical adoption of these needles remains poor. We did a systematic review and meta-analysis to compare patient outcomes after lumbar puncture with atraumatic needles and conventional needles. METHODS: In this systematic review and meta-analysis, we independently searched 13 databases with no language restrictions from inception to Aug 15, 2017, for randomised controlled trials comparing the use of atraumatic needles and conventional needles for any lumbar puncture indication. Randomised trials comparing atraumatic and conventional needles in which no dural puncture was done (epidural injections) or without a conventional needle control group were excluded. We screened studies and extracted data from published reports independently. The primary outcome of postdural-puncture headache incidence and additional safety and efficacy outcomes were assessed by random-effects and fixed-effects meta-analysis. This study is registered with the International Prospective Register of Systematic Reviews, number CRD42016047546. FINDINGS: We identified 20 241 reports; after exclusions, 110 trials done between 1989 and 2017 from 29 countries, including a total of 31 412 participants, were eligible for analysis. The incidence of postdural-puncture headache was significantly reduced from 11·0% (95% CI 9·1-13·3) in the conventional needle group to 4·2% (3·3-5·2) in the atraumatic group (relative risk 0·40, 95% CI 0·34-0·47, p<0·0001; I2=45·4%). Atraumatic needles were also associated with significant reductions in the need for intravenous fluid or controlled analgesia (0·44, 95% CI 0·29-0·64; p<0·0001), need for epidural blood patch (0·50, 0·33-0·75; p=0·001), any headache (0·50, 0·43-0·57; p<0·0001), mild headache (0·52, 0·38-0·70; p<0·0001), severe headache (0·41, 0·28-0·59; p<0·0001), nerve root irritation (0·71, 0·54-0·92; p=0·011), and hearing disturbance (0·25, 0·11-0·60; p=0·002). Success of lumbar puncture on first attempt, failure rate, mean number of attempts, and the incidence of traumatic tap and backache did not differ significantly between the two needle groups. Prespecified subgroup analyses of postdural-puncture headache revealed no interactions between needle type and patient age, sex, use of prophylactic intravenous fluid, needle gauge, patient position, indication for lumbar puncture, bed rest after puncture, or clinician specialty. These results were rated high-quality evidence as examined using the grading of recommendations assessment, development, and evaluation. INTERPRETATION: Among patients who had lumbar puncture, atraumatic needles were associated with a decrease in the incidence of postdural-puncture headache and in the need for patients to return to hospital for additional therapy, and had similar efficacy to conventional needles. These findings offer clinicians and stakeholders a comprehensive assessment and high-quality evidence for the safety and efficacy of atraumatic needles as a superior option for patients who require lumbar puncture. FUNDING: None.

Bacteria Getting into Shape: Genetic Determinants of E. coli Morphology.

Perturbation of cellular processes is a prevailing approach to understanding biology. To better understand the complicated biology that defines bacterial shape, a sensitive, high-content platform was developed to detect multiple morphological defect phenotypes using microscopy. We examined morphological phenotypes across the Escherichia coli K-12 deletion (Keio) collection at the mid-exponential growth phase, revealing 111 deletions perturbing shape. Interestingly, 64% of these were uncharacterized mutants, illustrating the complex nature of shape maintenance and regulation in bacteria. To understand the roles these genes play in defining morphology, 53 mutants with knockouts resulting in abnormal cell shape were crossed with the Keio collection in high throughput, generating 1,373 synthetic lethal interactions across 1.7 million double deletion mutants. This analysis yielded a highly populated interaction network spanning and linking multiple phenotypes, with a preponderance of interactions involved in transport, oxidation-reduction, and metabolic processes.IMPORTANCE Genetic perturbations of cellular functions are a prevailing approach to understanding cell systems, which are increasingly being practiced in very high throughput. Here, we report a high-content microscopy platform tailored to bacteria, which probes the impact of genetic mutation on cell morphology. This has particular utility in revealing elusive and subtle morphological phenotypes associated with blocks in nonessential cellular functions. We report 111 nonessential mutations impacting E. coli morphology, with nearly half of those genes being poorly annotated or uncharacterized. Further, these genes appear to be tightly linked to transport or redox processes within the cell. The screening platform is simple and low cost and is broadly applicable to any bacterial genomic library or chemical collection. Indeed, this is a powerful tool in understanding the biology behind bacterial shape.