Biology subjects

Alexander, L. M.

Publications and source records attributed to Alexander, L. M..

8 recordsLinked to original sources

bioRxiv2026

Compact adenine base editors to enable therapeutic rescue of Duchenne muscular dystrophy

Adenine base editors (ABEs) have emerged as a powerful gene-editing technology enabling precise and programmable adenine-to-guanine substitutions across the genome. However, their translation into in vivo therapeutics is limited by delivery challenges, as their size exceeds the packaging capacity of adeno-associated virus (AAV). Here, we report the discovery, structural characterization, and engineering of two compact, highly active ABEs built on novel deaminases and Cas9d nucleases, enabling all-in-one, single-vector AAV delivery. Applying these compact ABEs to Duchenne muscular dystrophy (DMD), we demonstrate efficient disruption of conserved splice-acceptor sites at dystrophin exons 45 and 51 in human skeletal muscle cells, enabling therapeutically relevant exon skipping. Together, these ABEs help expand the therapeutic reach of base editing towards diverse tissue types and disease targets.

bioengineering↗

bioRxiv2025

Comparative characterization of Cas12f orthologs reveals mechanistic features underlying enhanced genome editing efficiency

Miniature CRISPR-Cas12f nucleases are attractive candidates for therapeutic genome editing owing to their compact size and compatibility with adeno-associated virus (AAV) delivery. However, editing efficiencies in mammalian cells are lower than those of larger systems such as Cas12a and SpCas9. The extensive phylogenetic diversity of Cas12f suggests unexplored mechanistic variation with the potential for optimization. Here, we characterize a naturally occurring Cas12f ortholog discovered through metagenomics, Cas12f-MG119-28, which supports robust genome editing in human cells. Through structural, biochemical, and kinetic analyses, we compare Cas12f-MG119-28 with two recently described orthologs, Oscillibacter sp. Cas12f (OsCas12f) and Ruminiclostridium herbifermentans Cas12f (RhCas12f). These orthologs present divergent architectures and regulatory features governing PAM recognition, gRNA binding, dimerization, and DNA cleavage. Notably, Cas12f-MG119-28 achieves efficient R-loop formation via a stable dimer interface and a naturally optimized guide RNA. These discoveries elucidate key mechanistic determinants of Cas12f activity and may offer a framework for engineering compact genome editors with therapeutic potential.

biochemistry↗

bioRxiv2024

DNA targeting by compact Cas9d and its resurrected ancestor

The type II-A CRISPR effector SpCas9 has gained widespread popularity as an efficient and programmable genome editing tool. However, much remains to be known about novel compact variants that may overcome some limitations of current systems1,2. Recently, alternative CRISPR-Cas systems with highly compact nucleases capable of genome editing in mammalian cells have been discovered through metagenomic analysis of uncultivated microbes, including Cas9d (a type II-D CRISPR-Cas effector)3. Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains and facilitate DNA target recognition. We find that the sgRNA can be truncated by up to [~]25% yet still retain activity in vivo. We also show that despite preferentially targeting an NGG PAM, Cas9d exhibits a unique mechanism for PAM recognition. Finally, we identify the first Cas9d smaller than 800 amino acids exhibiting robust nuclease activity in mammalian cells. Using ancestral sequence reconstruction, we demonstrate that it is possible to generate compact nucleases capable of efficient genome editing by expanding the diversity of Cas9d families. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a molecular blueprint for future rational re-engineering of minimal RNA-guided DNA nucleases.

biochemistry↗

bioRxiv2020

Remote Digital Psychiatry: MindLogger for Mobile Mental Health Assessment and Therapy

BackgroundUniversal access to assessment and treatment of mental health and learning disorders remains a significant and unmet need. There is a vast number of people without access to care because of economic, geographic, and cultural barriers as well as limited availability of clinical experts who could help advance our understanding of mental health. ObjectiveTo create an open, configurable software platform to build clinical measures, mobile assessments, tasks, and interventions without programming expertise. Specifically, our primary requirements include: an administrator interface for creating and scheduling recurring and customized questionnaires where end users receive and respond to scheduled notifications via an iOS or Android app on a mobile device. Such a platform would help relieve overwhelmed health systems, and empower remote and disadvantaged subgroups in need of accurate and effective information, assessment, and care. This platform has potential to advance scientific research by supporting the collection of data with instruments tailored to specific scientific questions from large, distributed, and diverse populations. MethodsWe conducted a search for tools that satisfy the above requirements. We designed and developed a new software platform called "MindLogger" that exceeds the above requirements. To demonstrate the tools configurability, we built multiple "applets" (collections of activities) within the MindLogger mobile application and deployed several, including a comprehensive set of assessments underway in a large-scale, longitudinal, mental health study. ResultsOf the hundreds of products we researched, we found 10 that met our primary requirements above with 4 that support end-to-end encryption, 2 that enable restricted access to individual users data, 1 that provides open source software, and none that satisfy all three. We compared features related to information presentation and data capture capabilities, privacy and security, and access to the product, code, and data. We successfully built MindLogger mobile and web applications, as well as web browser-based tools for building and editing new applets and for administering them to end users. MindLogger has end-to-end encryption, enables restricted access, is open source, and supports a variety of data collection features. One applet is currently collecting data from children and adolescents in our mental health study, and other applets are in different stages of testing and deployment for use in clinical and research settings. ConclusionsWe have demonstrated the flexibility and applicability of the MindLogger platform through its deployment in a large-scale, longitudinal, mobile mental health study, and by building a variety of other mental health-related applets. With this release, we encourage a broad range of users to apply the MindLogger platform to create and test applets to advance health care and scientific research. We hope that increasing availability of applets designed to assess and administer interventions will facilitate access to health care in the general population.

neuroscience↗

bioRxiv2017

Balancing Strengths and Weaknesses in Dimensional Psychiatry

ObjectiveTo evaluate the feasibility and value of creating an extensible framework for psychiatric phenotyping that indexes both strengths and weaknesses of behavioral dimensions. The Extended Strengths and Weaknesses Assessment of Normal Behavior (E-SWAN) reconceptualizes each diagnostic criterion for selected DSM-5 disorders as a behavior, which can range from high (strengths) to low (weaknesses). Initial efforts have focused on Panic Disorder, Social Anxiety, Major Depression, and Disruptive Mood Dysregulation Disorder.\n\nMethodsData were collected from 523 participants (ages: 5-21 years old) in the Child Mind Institute Healthy Brain Network - an ongoing community-referred study. Parents completed each of the four E-SWAN scales and traditional unidirectional scales addressing the same disorders. Distributional properties, Item Response Theory Analysis (IRT) and Receiver Operating Characteristic (ROC) curves (for diagnostic prediction) were used to assess and compare the performance of E-SWAN and traditional scales.\n\nResultsIn contrast to the traditional scales, which exhibited truncated distributions, all four E-SWAN scales were found to have near-normal distributions. IRT analyses indicate the E-SWAN subscales provided reliable information about respondents throughout the population distribution; in contrast, traditional scales only provided reliable information about respondents at the high end of the distribution. Predictive value for DSM-5 diagnoses was comparable to prior scales.\n\nConclusionE-SWAN bidirectional scales can capture the full spectrum of the population distribution for DSM disorders. The additional information provided can better inform examination of inter-individual variation in population studies, as well as facilitate the identification of factors related to resiliency in clinical samples.

neuroscience↗

bioRxiv2017

A multi-modal approach to decomposing standard neuropsychological test performance: Symbol Search

Neuropsychological test batteries provide normed assessments of cognitive performance across multiple functional domains. Although each test emphasizes a certain component of cognition, a poor score can reflect many possible processing deficits. Here we explore the use of simultaneous eye tracking and EEG to decompose test performance into interpretable, components of cognitive processing. We examine the specific case of Symbol Search, a \"processing speed\" subtest of the WISC, which involves searching for the presence of either of two target symbols among five search symbols. To characterize the signatures of effective performance of the test, we asked 26 healthy adults to perform a computerized version of it while recording continuous EEG and eye tracking. We first established basic gaze-shifting patterns in the task, such as more frequent and prolonged fixation of each target than each search symbol, and longer search symbol fixations and overall trial duration for target-absent trials. We then entered multiple such metrics into a least absolute shrinkage and selection operator (LASSO) analysis, which revealed that short trial completion times were mainly predicted by longer initial fixations on the targets and fewer subsequent confirmatory saccades directed back to the targets. Further, the tendency to make confirmatory saccades was associated with stronger gamma-amplitude modulation by mid-frontal theta-phase in the EEG during initial target symbol encoding. Taken together, these findings indicate that efficient Symbol Search performance depends more on effective memory encoding than on general \"processing speed\".

neuroscience↗

bioRxiv2017

The Healthy Brain Network Biobank: An open resource for transdiagnostic research in pediatric mental health and learning disorders

Technological and methodological innovations are equipping researchers with unprecedented capabilities for detecting and characterizing pathologic processes in the developing human brain. As a result, ambitions to achieve clinically useful tools to assist in the diagnosis and management of mental health and learning disorders are gaining momentum. To this end, it is critical to accrue large-scale multimodal datasets that capture a broad range of commonly encountered clinical psychopathology. The Child Mind Institute has launched the Healthy Brain Network (HBN), an ongoing initiative focused on creating and sharing a biobank of data from 10,000 New York area participants (ages 5-21). The HBN Biobank houses data about psychiatric, behavioral, cognitive, and lifestyle phenotypes, as well as multimodal brain imaging (resting and naturalistic viewing fMRI, diffusion MRI, morphometric MRI), electroencephalography, eye-tracking, voice and video recordings, genetics, and actigraphy. Here, we present the rationale, design and implementation of HBN protocols. We describe the first data release (n = 664) and the potential of the biobank to advance related areas (e.g., biophysical modeling, voice analysis).

neuroscience↗

bioRxiv2016

A Resource for Assessing Information Processing in the Developing Brain Using EEG and Eye Tracking

We present a dataset combining electrophysiology and eye tracking intended as a resource for the investigation of information processing in the developing brain. The dataset includes high-density task-based and task-free EEG, eye tracking, and cognitive and behavioral data collected from 126 individuals (ages: 6-44). The task battery spans both the simple/complex and passive/active dimensions to cover a range of approaches prevalent in modern cognitive neuroscience. The active task paradigms facilitate principled deconstruction of core components of task performance in the developing brain, whereas the passive paradigms permit the examination of intrinsic functional network activity during varying amounts of external stimulation. Alongside these neurophysiological data, we include an abbreviated cognitive test battery and questionnaire-based measures of psychiatric functioning. We hope that this dataset will lead to the development of novel assays of neural processes fundamental to information processing, which can be used to index healthy brain development as well as detect pathologic processes.

neuroscience↗