Biology subjects

Lee, M.

Publications and source records attributed to Lee, M..

At least 109 records · Page 6Linked to original sources

bioRxiv2024

Detection of genome structural variation in normal cells and tissues by single molecule sequencing

Rare genome structural-variants (SVs) in somatic cells and tissues are difficult to measure in bulk DNA because library preparation can create chimeric junctions that mimic authentic events. Based on our previous Single-Molecule Mutation sequencing (SMM-seq) assay, we developed a new method, SMM-SV-seq, which combines a chimera-suppressing library preparation with duplex confirmation of breakpoint-supporting molecules. Using libraries from mixtures of human and Drosophila DNA, we show a remaining artifact rate of 1.52 +/- 0.53 detectable cross-species chimeras per billion bases, providing an empirical background for correcting breakpoint frequencies. In a benchmark of evaluable homozygous germline deletions in the GM24385 cell line, SMM-SV-seq achieved 81.4% precision and 85.7% recall, exceeding the precision of established short-read callers while maintaining comparable recall. The assay detected dose-associated increases in SV-breakpoint frequencies in cells treated with bleomycin, a known clastogen. Biological relevance was demonstrated by a significantly greater irradiation-associated increase in translocation-like breakpoint frequencies in the BRCA1 185delAG-heterozygous mammary epithelial clone than in its isogenic wild-type control. These results establish SMM-SV-seq as a practical assay for calibrated, duplex-confirmed detection of rare somatic SV breakpoints directly in bulk DNA from non-clonal cell populations.

genomics↗

bioRxiv2024

Conformations of a Low-Complexity Protein in Homogeneous and Phase-Separated Frozen Solutions

Solutions of the intrinsically disordered, low-complexity domain of the FUS protein (FUS-LC) undergo liquid-liquid phase separation (LLPS) below temperatures TLLPS in the 20-40{degrees} C range. To investigate whether local conformational distributions are detectably different in the homogeneous and phase-separated states of FUS-LC, we performed solid state nuclear magnetic resonance (ssNMR) measurements on solutions that were frozen on sub-millisecond time scales after equilibration at temperatures well above (50{degrees} C) or well below (4{degrees} C) TLLPS. Measurements were performed at 25 K with signal enhancements from dynamic nuclear polarization. Crosspeak patterns in two-dimensional (2D) ssNMR spectra of rapidly frozen solutions in which FUS-LC was uniformly 15N,13C-labeled were found to be nearly identical for the two states. Similar results were obtained for solutions in which FUS-LC was labeled only at Thr, Tyr, and Gly residues, as well as solutions of a FUS construct in which five specific residues were labeled by ligation of synthetic and recombinant fragments. These experiments show that local conformational distributions are nearly the same in the homogeneous and phase-separated solutions, despite the much greater protein concentrations and more abundant intermolecular interactions within phase-separated, protein-rich "droplets". Comparison of the experimental results with simulations of the sensitivity of 2D crosspeak patterns to an enhanced population of {beta}-strand-like conformations suggests that changes in conformational distributions are no larger than 5-10%. Statement of SignificanceLiquid-liquid phase separation (LLPS) in solutions of proteins with intrinsically disordered domains has attracted recent attention because of its relevance to multiple biological processes and its inherent interest from the standpoint of protein biophysics. The high protein concentrations and abundant intermolecular interactions within protein-rich, phase-separated "droplets" suggests that conformational distributions of intrinsically disordered proteins may differ in homogeneous and phase-separated solutions. To investigate whether detectable differences exist, we performed experiments on the low-complexity domain of the FUS protein (FUS-LC) in which FUS-LC solutions were first equilibrated at temperatures well above or well below their LLPS transition temperatures, then rapidly frozen and examined at very low temperatures by solid state nuclear magnetic resonance (ssNMR) spectroscopy. The ssNMR data for homogeneous and phase-separated frozen solutions of FUS-LC were found to be nearly identical, showing that LLPS is not accompanied by substantial changes in the local conformational distributions of this intrinsically disordered protein.

biophysics↗

bioRxiv2024

Cryopreservation of neuroectoderm on a pillar plate and in situ differentiation into human brain organoids

Cryopreservation in cryovials extends cell storage at low temperatures, and advances in organoid cryopreservation improve reproducibility and reduce generation time. However, cryopreserving human organoids presents challenges due to the limited diffusion of cryoprotective agents (CPAs) into the organoid core and the potential toxicity of these agents. To overcome these obstacles, we developed a cryopreservation technique using a pillar plate platform. To illustrate cryopreservation application to human brain organoids (HBOs), early-stage HBOs were produced by differentiating induced pluripotent stem cells (iPSCs) into neuroectoderm (NEs) in an ultralow atachement (ULA) 384-well plate. These NEs were transferred and encapsulated in Matrigel on the pillar plate. The early-stage HBOs on the pillar plate were exposed to four commercially available CPAs, including PSC cryopreservation kit, CryoStor CS10, 3dGRO, and 10% DMSO, before being frozen overnight at -80{degrees}C and subsequently stored in a liquid nitrogen dewar. We examined the impact of CPA type, organoid size, and CPA exposure duration on cell viability post-thaw. Additionally, the differentiation of early-stage HBOs on the pillar plate was assessed using RT-qPCR and immunofluorescence staining. The PSC cryopreservation kit proved to be the least toxic for preserving these HBOs on the pillar plate. Notably, smaller HBOs showed higher cell viability post-cryopreservation than larger ones. An incubation period of 80 minutes with the PSC kit was essential to ensure optimal CPA diffusion into HBOs with a diameter of 400 - 600 {micro}m. These cryopreserved early-stage HBOs successfully matured over 30 days, exhibiting gene expression patterns akin to non-cryopreserved HBOs. The cryopreserved early-stage HBOs on the pillar plate maintained high viability after thawing and successfully differentiated into mature HBOs. This on-chip cryopreservation method could extend to other small organoids, by integrating cryopreservation, thawing, culturing, staining, rinsing, and imaging processes within a single system, thereby preserving the 3D structure of the organoids.

bioengineering↗

bioRxiv2024

DrugDiff - small molecule diffusion model with flexible guidance towards molecular properties

With the cost/yield-ratio of drug development becoming increasingly unfavourable, recent work has explored machine learning to accelerate early stages of the development process. Given the current success of deep generative models across domains, we here investigated their application to the property-based proposal of new small molecules for drug development. Specifically, we trained a latent diffusion model -- DrugDiff -- paired with predictor guidance to generate novel compounds with a variety of desired molecular properties. The architecture was designed to be highly flexible and easily adaptable to future scenarios. Our experiments showed successful generation of unique, diverse and novel small molecules with targeted properties. The code is available at https://github.com/MarieOestreich/DrugDiff.

bioinformatics↗

bioRxiv2024

The role of YAP/TAZ signaling in dendritic cell-mediated pathogenesis of insulin resistance and non-alcoholic fatty liver disease

Obesity and insulin resistance (IR) are global health challenges linked to metabolic diseases, such as type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). High-caloric intake, which is associated to NAFLD, induces adipocyte hypertrophy and inflammation, triggering dendritic cell (DC) activation and systemic inflammation. DC exacerbate inflammation by promoting pro-inflammatory responses, aggravating IR and NAFLD progression. NAFLD is characterized by liver fibrosis, which alters tissue stiffness that can trigger mechanosensing pathways such as the Hippo pathway in immune cell types. In this work we explored the roles of key mediators of the Hippo pathway, YAP and TAZ, in DCs within the context of liver fibrosis, obesity and IR, using a model of NAFLD induced by feeding a high fat high sucrose diet. Our findings indicate that specific deletion of YAP and/or TAZ in DCs had minimal impact on IR development and metabolic tissue inflammation. We conclude that YAP and TAZ have limited and possibly redundant roles in the immune pathophysiology of NAFLD and IR.

immunology↗

bioRxiv2024

HIV-1-envelope trimer transitions from prefusion-closed to CD4-bound-open conformations through an occluded-intermediate state

HIV-1 infection is initiated by the interaction between the gp120 subunit in the envelope (Env) trimer and the cellular receptor CD4 on host cells. This interaction induces substantial structural rearrangement of the Env trimer. Currently, static structural information for prefusion-closed trimers, CD4-bound prefusion-open trimers, and various antibody-bound trimers is available. However, dynamic features between these static states (e.g., transition structures) are not well understood. Here, we investigate the full transition pathway of a site specifically glycosylated Env trimer between prefusion-closed and CD4-bound-open conformations by collective molecular dynamics and single-molecule Forster resonance energy transfer (smFRET). Our investigations reveal and confirm important features of the transition pathway, including movement of variable loops to generate a glycan hole at the trimer apex and formation or rearrangements of -helices and {beta}-strands. Notably, by comparing the transition pathway to known Env-structures, we uncover evidence for a transition intermediate, with four antibodies, Ab1303, Ab1573, b12, and DH851.3, recognizing this intermediate. Each of these four antibodies induce population shifts of Env to occupy a newly observed smFRET state: the "occluded-intermediate" state. We propose this occluded-intermediate state to be both a prevalent state of Env and an on-path conformation between prefusion-closed and CD4-bound-open states, previously overlooked in smFRET analyses.

bioinformatics↗

bioRxiv2024

The chemical chaperone 4-phenylbutyric acid rescues molecular cell defects of COL3A1 mutations that cause vascular Ehlers Danlos Syndrome

PurposeVascular Ehlers Danlos Syndrome (vEDS) is a connective tissue disorder caused by COL3A1 mutations for which there are no treatments due to a limited understanding of underlying mechanisms. We aimed to address this critical knowledge gap, focusing on collagen folding, to establish if targeting protein folding represents a potential therapeutic approach. MethodsWe performed a mechanistic analysis of two novel COL3A1 glycine mutations, G189S and G906R, using primary patient fibroblast cultures, and performed pre-clinical proof-of-concept treatments using FDA-approved chemical chaperones targeting protein folding and/or degradation. ResultsCOL3A1 mutations caused secretion of misfolded collagen III and intracellular collagen retention, leading to matrix defects and endoplasmic reticulum (ER) stress, with increased severity for the more C-terminal mutation. Promoting ER protein folding capacity through the chemical chaperone 4-phenylbutyric acid rescued the ER stress, thermostability of secreted collagen, matrix defects and apoptosis. Optimising treatment duration and dosage helped overcome allele-dependent treatment efficacy. In contrast, protein degradation alone or combined with targeting protein folding did not increase efficacy. ConclusionER stress is a molecular mechanism in vEDS that can be influenced by the position of COL3A1 mutation, and promoting protein folding is a putative mechanism-based therapeutic approach that can rescue intra- and extracellular defects.

genetics↗

bioRxiv2024

Vaccine-elicited and naturally elicited antibodies differ in their recognition of the HIV-1 fusion peptide

Broadly neutralizing antibodies have been proposed as templates for HIV-vaccine design, but it has been unclear how similar vaccine-elicited antibodies are to their naturally elicited templates. To provide insight, here we compare the recognition of naturally elicited and vaccine-elicited antibodies targeting the HIV-1-fusion peptide, which comprises envelope (Env) residues 512-526, with the most common sequence being AVGIGAVFLGFLGAA. Naturally elicited antibodies bound peptides with negative-charge substitutions around residues 517-520 substantially better than the most common sequence, despite these substitutions rarely appearing in HIV; by contrast, vaccine-elicited antibodies were less tolerant of sequence variation, with no substitution of residues 512-516 showing increased binding. Molecular dynamics analysis and cryo-EM structure of the naturally elicited ACS202 antibody in complex with HIV-Env trimer with A517E suggested enhanced binding to result from electrostatic interactions with positively charged antibody residues. Overall, vaccine-elicited antibodies appeared to be more fully optimized to bind the most common fusion peptide sequence. HIGHLIGHTSPeptide substitution scan reveals naturally elicited antibodies against fusion peptide (FP) can bind select non-canonical FP sequences with high affinity. Peptide substitution scan data for FP antibodies correlates significantly with their differential selection indicating variation in binding relates to neutralization tolerance. Structure and energetic analysis of naturally elicited ACS202 with HIV-Env trimer reveals basis for improved recognition of A517E mutant. Atomic level interactions from MD simulation analysis corroborate trends observed with peptide substitutions. verall, peptide substitution scans reveal vaccine-elicited antibodies against FP to be less permissive to FP-sequence variability than naturally elicited antibodies.

immunology↗

bioRxiv2024

Missense Mutant Gain-of-Function Causes Inverted Formin 2 (INF2)-Related Focal Segmental Glomerulosclerosis (FSGS)

Inverted formin-2 (INF2) gene mutations are among the most common causes of genetic focal segmental glomerulosclerosis (FSGS) with or without Charcot-Marie-Tooth (CMT) disease. Recent studies suggest that INF2, through its effects on actin and microtubule arrangement, can regulate processes including vesicle trafficking, cell adhesion, mitochondrial calcium uptake, mitochondrial fission, and T-cell polarization. Despite roles for INF2 in multiple cellular processes, neither the human pathogenic R218Q INF2 point mutation nor the INF2 knock-out allele is sufficient to cause disease in mice. This discrepancy challenges our efforts to explain the disease mechanism, as the link between INF2-related processes, podocyte structure, disease inheritance pattern, and their clinical presentation remains enigmatic. Here, we compared the kidney responses to puromycin aminonucleoside (PAN) induced injury between R218Q INF2 point mutant knock-in and INF2 knock-out mouse models and show that R218Q INF2 mice are susceptible to developing proteinuria and FSGS. This contrasts with INF2 knock-out mice, which show only a minimal kidney phenotype. Co-localization and co-immunoprecipitation analysis of wild-type and mutant INF2 coupled with measurements of cellular actin content revealed that the R218Q INF2 point mutation confers a gain-of-function effect by altering the actin cytoskeleton, facilitated in part by alterations in INF2 localization. Differential analysis of RNA expression in PAN-stressed heterozygous R218Q INF2 point-mutant and heterozygous INF2 knock-out mouse glomeruli showed that the adhesion and mitochondria-related pathways were significantly enriched in the disease condition. Mouse podocytes with R218Q INF2, and an INF2-mutant human patients kidney organoid-derived podocytes with an S186P INF2 mutation, recapitulate the defective adhesion and mitochondria phenotypes. These results link INF2-regulated cellular processes to the onset and progression of glomerular disease. Thus, our data demonstrate that gain-of-function mechanisms drive INF2-related FSGS and explain the autosomal dominant inheritance pattern of this disease.

genetics↗

bioRxiv2024

Mechanical Rejuvenation of Mesenchymal Stem Cells from Aged Patients

Mesenchymal stem cells (MSC) are an appealing therapeutic cell type for many diseases. However, patients with poor health or advanced age often have MSCs with poor regenerative properties. A major limiter of MSC therapies is cellular senescence, which is marked by limited proliferation capability, diminished multipotency, and reduced regenerative properties. In this work, we explored the ability of applied mechanical forces to reduce cellular senescence in MSCs. Our studies revealed that mechanical conditioning caused a lasting enhancement in proliferation, overall cell culture expansion potential, multipotency, and a reduction of senescence in MSCs from aged donors. Mechanistic studies suggested that these functional enhancements were mediated by oxidative stress and DNA damage repair signaling with mechanical load altering the expression of proteins of the sirtuin pathway, the DNA damage repair protein ATM, and antioxidant proteins. In addition, our results suggest a biophysical mechanism in which mechanical stretch leads to improved recognition of damaged DNA in the nucleus. Analysis of the cells through RNA-seq and ATAC-seq, demonstrated that mechanical loading alters the cells genetic landscape to cause broad shifts in transcriptomic patterns that related to senescence. Overall, our results demonstrate that mechanical conditioning can rejuvenate mesenchymal stem cells derived from aged patients and improve their potential as a therapeutic cell type. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/597781v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@a479d4org.highwire.dtl.DTLVardef@3a728eorg.highwire.dtl.DTLVardef@136dc3dorg.highwire.dtl.DTLVardef@b54d51_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Characterization of Viroplasm-Like Structures by Co-Expression of NSP5 and NSP2 Across Rotavirus Species A to J

Rotaviruses (RV) are classified into nine species, A-C and D-J, with species A being the most studied. In rotavirus of species A (RVA), replication occurs in viroplasms, which are cytosolic globular inclusions primarily composed of the proteins NSP5, NSP2, and VP2. The co-expression of NSP5 with either NSP2 or VP2 leads to the formation of viroplasm-like structures (VLS). Although morphologically identical to viroplasms, VLSs cannot replicate, but they serve as excellent simplified tools for studying complex viroplasms. There is a knowledge gap regarding viroplasms of non-RVA species due to a lack of research tools, such as specific antibodies and tissue culture systems. In this study, we explored the ability of NSP5 and NSP2 from non-RVA species to form VLSs. The co-expression of these two proteins led to globular VLSs in RV species A, B, D, F, G, and I, while RVC formed filamentous VLSs. The co-expression of NSP5 and NSP2 of RV species H and J did not result in VLS formation. Interestingly, NSP5 of all RV species self-oligomerizes, with the ordered C-terminal region, termed the tail, being necessary for self-oligomerization of RV species A-C and G-J. Except for NSP5 from species J, all NSP5 bound with their respective NSP2. We also found that interspecies VLS are formed between closely related RV species B with G and D with F. Additionally, VLS from RVH and RVJ formed when the tail of NSP5 RVH and RVJ was replaced by the tail of NSP5 from RVA and co-expressed with their respective NSP2. ImportanceRotaviruses (RV) are classified into nine species, A-D and F-J, infecting mammals and birds. Due to the lack of research tools, all cumulative knowledge on RV replication is based on RV species A (RVA). The RV replication compartments are globular cytosolic structures named viroplasms, which have only been identified in RV species A. In this study, we examined the formation of viroplasm-like structures (VLS) by the expression of NSP5 with NSP2 across RV species A to J. Globular VLSs formed for RV species A, B, D, F, G, and I, while RV species C formed filamentous structures. The RV species H and J did not form VLS with NSP5 and NSP2. Similar to RVA, NSP5 self-oligomerizes in all RV species, which is a requirement for VLS formation. This study provides basic knowledge of the non-RVA replication mechanisms, which could help develop strategies to halt virus infection across RV species.

microbiology↗

bioRxiv2024

Participatory science methods to monitor water quality and ground truth remote sensing of the Chesapeake Bay

Measurements by volunteer scientists using participatory science methods in combination with high resolution remote sensing can improve our ability to monitor water quality changes in highly vulnerable and economically valuable nearshore and estuarine habitats. In the Chesapeake Bay (USA), tidal tributaries are a focus of watershed and shoreline management efforts to improve water quality. The Chesapeake Water Watch program seeks to enhance the monitoring of tributaries by developing and testing methods for volunteer scientists to easily measure chlorophyll, turbidity, and colored dissolved organic matter (CDOM) to inform Bay stakeholders and improve algorithms for analogous remote sensing (RS) products. In the program, trained volunteers have measured surface turbidity using a smartphone app, HydroColor, calibrated with a photographers gray card. In vivo chlorophyll and CDOM fluorescence were assessed in surface samples with hand-held fluorometers (Aquafluor) located at sample processing "hubs" where volunteers drop-off samples for same day processing. In validation samples, HydroColor turbidity and Aquafluor in vivo chlorophyll and CDOM fluorescence were linearly related to standard analytical measures of turbidity, chlorophyll and CDOM, respectively, with R2 values ranging from 0.65 to 0.85. These methods are being used for both repeat sampling at fixed sites of interest and ad-hoc "blitzes" to synoptically sample tributaries all around the Bay in coordination with satellite overpasses. All data is accessible on a public database (serc.fieldscope.org) and can be a resource to monitor long-term trends in the tidal tributaries as well as detect and diagnose causes of events of concern such as algal blooms and storm-induced reductions in water clarity.

ecology↗

bioRxiv2024

Ancient eukaryotic protein interactions illuminate modern genetic traits and disorders

All eukaryotes share a single-celled ancestor from [~]1.5-1.8 billion years ago, the Last Eukaryotic Common Ancestor (LECA). Roughly half of gene families found in modern eukaryotes were already present in LECA, forming molecular systems that continue to influence genetic diseases and traits today. To investigate these systems, we compared genes across 156 organisms to define a core set of protein-coding gene families likely present in LECA, with a quarter remaining uncharacterized. Integrating >26,000 mass spectrometry proteomics analyses from 31 species, we inferred higher-order complexes among these ancient proteins. This reconstructed interactome reveals both established and novel assemblies, offering a biochemical snapshot of LECAs organization. Finally, by exploring these ancient protein interactions, we found new human gene-disease associations for bone density and congenital birth defects, illustrating the value of ancestral protein networks for modern functional genetics.

evolutionary biology↗

bioRxiv2024

Realigning representational drift in mouse visual cortex by flexible brain-machine interfaces

The ability to stably decode brain activity is crucial for brain-machine interfaces (BMIs), which are often compromised by recording instability due to immune responses and probe drifting. In addition, many brain regions undergo intrinsic dynamics such as "representational drift", in which neural activities associated with stable sensation and action continually change over time. In this study, we employed tissue-like flexible electrode arrays for recording visual stimulus-dependent single-unit action potentials in the mouse visual cortex. The flexible electrode array enabled us to record action potentials from the same neurons over extended periods under visual stimuli, allowing us to characterize the representational drift during these stimuli. With this approach, we tested hypotheses about the origins and mechanisms of representational drift, tracked latent dynamics transformation, and modeled these dynamics with affine transformation. Our findings enabled the construction of a single, long-term stable, high-performance visual information decoder that accounts for representational drift, potentiating chronically stable flexible BMIs in brain regions experiencing representational drifts.

bioengineering↗

bioRxiv2024

Initiation of Lumen Formation from Junctions via Differential Actomyosin Contractility Regulated by Dynamic Recruitment of Rasip1

Vascular lumens are essential for the dispersal of nutrients, oxygen, and immune cells throughout the body. De novo lumen formation necessitates the precise segregation of junctional proteins from apical surfaces. Direct visualization of this process and a comprehensive understanding on how the apical and junctional compartments are segregated and established remain elusive, due to the lack of visualizable systems, proper reporters and manipulative tools. Using zebrafish as an experimental model, we have established a wealth of molecular reporters, photo-convertible and optogenetic tools and combined them with genetic analyses to study the establishment of apical domains from pre-apical junctional complexes. Our study identified Rasip1 as one of the earliest apical proteins recruited, peripheralizing cortical contractility at the junctional patches by inhibiting NMII at the center, thereby initiating the outward flow of junctional complexes. After the establishment of the apical compartments, Rasip1 shuttles between junctions and the apical compartments to the high-tension sites and tunes down the local hyper contractility, restricting Cdh5 to junctions by suppressing apical contractility. Above all, the formation of de novo lumens and maintenance of junctional integrity depend on the precise control of contractility within the apical compartments and junctions, orchestrated by the dynamic recruitment of Rasip1.

developmental biology↗

bioRxiv2024

Structural Determinants of Vibrio cholerae FeoB Nucleotide Promiscuity

Ferrous iron (Fe2+) is required for the growth and virulence of many pathogenic bacteria, including Vibrio cholerae (Vc), the causative agent of the disease cholera. For this bacterium, Feo is the primary system that transports Fe2+ into the cytosol. FeoB, the main component of this system, is regulated by a soluble cytosolic domain termed NFeoB. Recent reanalysis has shown that NFeoBs can be classified as either GTP-specific or NTP-promiscuous, but the structural and mechanistic bases for these differences were not known. To explore this intriguing property of FeoB, we solved the X-ray crystal structures of VcNFeoB in both the apo and GDP-bound forms. Surprisingly, this promiscuous NTPase displayed a canonical NFeoB G-protein fold like GTP-specific NFeoBs. Using structural bioinformatics, we hypothesized that residues surrounding the nucleobase could be important for both nucleotide affinity and specificity. We then solved the X-ray crystal structures of N150T VcNFeoB in the apo and GDP-bound forms to reveal H-bonding differences surround the guanine nucleobase. Interestingly, isothermal titration calorimetry revealed similar binding thermodynamics of the WT and N150T proteins to guanine nucleotides, while the behavior in the presence of adenine nucleotides was dramatically different. AlphaFold models of VcNFeoB in the presence of ADP and ATP showed important conformational changes that contribute to nucleotide specificity among FeoBs. Combined, these results provide a structural framework for understanding FeoB nucleotide promiscuity, which could be an adaptive measure utilized by pathogens to ensure adequate levels of intracellular iron across multiple metabolic landscapes.

biochemistry↗

bioRxiv2024

Efficient DNA base editing via an optimized DYW-like deaminase

CRISPR-based cytosine base editors enable precise genome editing without inducing double-stranded DNA breaks, yet traditionally depend on a limited selection of deaminases from the APOBEC/AID or TadA families. Here, we introduce SsCBE, a novel CRISPR-based cytosine base editor utilizing SsdAtox, a DYW-like deaminase derived from the toxin of Pseudomonas syringae. Strategic engineering of SsdAtox has led to remarkable improvements in the base editing efficiency (by up to 8.4-fold) and specificity for SsCBE, while concurrently reducing cytotoxicity. Exhibiting exceptional versatility, SsCBE was delivered and efficiently applied using diverse delivery methods, including the engineered virus-like particles (eVLPs). Its application has enabled targeted cytosine base editing in mouse zygotes and pioneering edits in mitochondrial DNA. The advent of SsCBE marks a significant advancement in the CRISPR toolkit, providing a versatile tool for advanced research and therapeutic strategies.

molecular biology↗

bioRxiv2024

Negative Selection Allows DNA Mismatch Repair-Deficient Mouse Fibroblasts In Vitro to Tolerate High Levels of Somatic Mutations

Substantial numbers of somatic mutations have been found to accumulate with age in different human tissues. Clonal cellular amplification of some of these mutations can cause cancer and other diseases. However, it is as yet unclear if and to what extent an increased burden of random mutations can affect cellular function without clonal amplification. We tested this in cell culture, which avoids the limitation that an increased mutation burden in vivo typically leads to cancer. We performed single-cell whole-genome sequencing of primary fibroblasts from DNA mismatch repair (MMR) deficient Msh2-/- mice and littermate control animals after long-term passaging. Apart from analyzing somatic mutation burden we analyzed clonality, mutational signatures, and hotspots in the genome, characterizing the complete landscape of somatic mutagenesis in normal and MMR-deficient mouse primary fibroblasts during passaging. While growth rate of Msh2-/-fibroblasts was not significantly different from the controls, the number of de novo single-nucleotide variants (SNVs) increased linearly up until at least 30,000 SNVs per cell, with the frequency of small insertions and deletions (INDELs) plateauing in the Msh2-/- fibroblasts to about 10,000 INDELS per cell. We provide evidence for negative selection and large-scale mutation-driven population changes, including significant clonal expansion of preexisting mutations and widespread cell-strain-specific hotspots. Overall, our results provide evidence that increased somatic mutation burden drives significant cell evolutionary changes in a dynamic cell culture system without significant effects on growth. Since similar selection processes against mutations preventing organ and tissue dysfunction during aging are difficult to envision, these results suggest that increased somatic mutation burden can play a causal role in aging and diseases other than cancer.

genomics↗