Biology subjects

Singh, K.

Publications and source records attributed to Singh, K..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

PI(3)P signaling regulates endosomal flux underlying developmental synaptic remodeling via Rab4

Rab4 GTPase, essential for endosomal sorting and trafficking, is implicated in synaptic atrophy and dementia. To uncover the underlying mechanism, we studied the correlation between Rab4 vesicle transport in axons and episodic remodeling of synapses in the central nervous system (CNS) of Drosophila larvae. We found that synapse-bound traffic and presynaptic enrichment of Rab4 vesicles increase during the programmed, transient contraction of synapses in the ventral neuropil region at a specific larval stage. This coincides with the episodic activation of insulin and Vps34-mediated signaling, which elevates phosphatidylinositol-3-phosphate levels on Rab4 vesicles. The presence of this phospholipid on Rab4-associated vesicles recruit a PX-domain-containing motor protein, Klp98A, accelerating synapse-directed traffic. This, in turn, increases presynaptic enrichment of Rab4 during the developmentally programmed synapse contraction phase. Our findings elucidate the molecular mechanism that regulates developmental synaptic plasticity in the CNS via insulin signaling and directed axonal transport of endosomes. Author summaryHealthy brain function relies on the growth and remodeling of synaptic connections between nerve cells. This process involves carefully orchestrated movements of proteins and membranes inside the neurites. For instance, endosome movement in the nerve cell extensions helps guide neuronal growth, recycle parts of the synapse, and alter signal transmissions between neurons. Rab GTPases, a group of small G-proteins, are crucial for these processes. Here, we aimed to understand what triggers these processes and how the supply chain within nerve cell extensions is connected to changes at the synapse. In fruit fly larvae, we observed that the movement of Rab4 vesicles in the nerve cell extensions is inversely related to gross synapse content. A further examination revealed that onset of insulin signaling accelerates the Rab4 vesicles towards synapse through a lipid kinase called Vps34, which makes a signaling lipid called phospatidylinnositol-3-phosphate. Higher levels this lipid molecule on Rab4 vesicles attract a motor protein, Klp98A, speeding up their movement during the synaptic contraction phase. Our findings reveal a new mechanism that regulates Rab4 vesicle transport and its impact on synapse stability. The study further suggests potential targets for controlling age-related dementia likely to be caused by excessive insulin signaling in neurons.

neuroscience↗

bioRxiv2025

Th1 and Th2 cytokines profile of serum samples from dengue positive cases from Northeast India

BackgroundDengue fever is a fast-spreading acute arboviral infection transmitted by a human-Aedes mosquito cycle. Northeastern India is an endemic zone for dengue; regular outbreaks have been reported. However, molecular details of the virus infection in the host have not been established. Studies on dengue immunopathogenesis have primarily utilized data from American and Southeast Asian regions, with insufficient representation from India. ObjectiveThe present study aimed to characterize the Th1/Th2 cytokine expression profiles from dengue cases in different parts of northeastern India. Statistical analysis cum ResultsAmong the panel of Th1/Th2 cytokines, IFN-{gamma}, IL-2, IL-4, IL-5, IL-10, IL-13, and TNF- cytokines are found to be higher in dengue-infected patients. Anti-inflammatory cytokines are somehow regulated differently in male-female patients. ConclusionThis clinical study on dengue patient samples for cytokine profiling revealed distinct cytokine patterns and their regulation, but further investigation is needed to correlate with disease severity to identify potential biomarkers for disease progression.

microbiology↗

bioRxiv2025

Cigarette smoke and biological age induce degenerative heterogeneity in retinalpigment epithelium

Environmental exposure such as cigarette smoke induces epigenetic changes that can induce degenerative heterogeneity and accelerate aging. In early age-related macular degeneration (AMD), the leading worldwide cause of blindness among the elderly, retinal pigment epithelial (RPE) cell heterogeneity is a key change. Since smoking is the strongest environmental risk factor for AMD, we hypothesized that cigarette smoke induces degenerative RPE heterogeneity through epigenetic changes that are distinct from aging, and that with aging, the RPE becomes vulnerable to cigarette smoke insult. We administered cigarette smoke condensate (CSC) intravitreally to young and aged mice and performed snRNA-seq and snATAC-seq on the RPE/choroid. This analysis identified separate cell clusters corresponding to healthy and abnormal, dedifferentiated RPE in both aged vehicle-treated and young CSC-treated mice. The dedifferentiated RPE were characterized by a global decrease in chromatin accessibility and decreased expression of genes in functional categories that were linked to hallmarks of aging. Notably, young, dedifferentiated RPE also exhibited a compensatory upregulation of hallmarks of aging-related genes, specifically those related to mitochondrial function and proteostasis. In contrast, aged dedifferentiated RPE did not express these compensatory changes, and did not survive CSC treatment, as experimentally verified with TUNEL labeling. These changes are relevant to early AMD because we identified through scRNA-seq, similar dedifferentiated and healthy macular RPE clusters in a donor who smoked and another with early AMD, but not from a nonsmoker. Degenerative cellular heterogeneity can include an abnormal cluster that jeopardizes cell survival and may represent an additional hallmark of ocular aging.

neuroscience↗

bioRxiv2025

Biphasic inflammation control by dedifferentiated fibroblasts enables axon regeneration after spinal cord injury in zebrafish

Fibrosis and persistent inflammation are interconnected processes that inhibit axon regeneration in the mammalian central nervous system (CNS). In zebrafish, by contrast, fibroblast-derived extracellular matrix deposition and inflammation facilitate regeneration. However, the regulatory cross-talk between fibroblasts and the innate immune system in the regenerating CNS is not understood. Here, we show that zebrafish fibroblasts possess a dual role in inducing and subsequently resolving inflammation, which are both essential for regeneration. We identify a transient, injury-specific cthrc1a+ fibroblast state with an inflammation-associated, less differentiated, and non-fibrotic profile. Induction of this fibroblast state precedes and contributes to the initiation of the inflammatory response. At the peak of neutrophil influx, cthrc1a+ fibroblasts coordinate the resolution of inflammation. Disruption of these inflammation dynamics inhibits axon regeneration and alters the mechano-structural properties of the lesion environment. This establishes the biphasic inflammation control by dedifferentiated fibroblasts as a pivotal mechanism for CNS regeneration. ONE SENTENCE SUMMARYDedifferentiated fibroblasts sequentially induce and resolve neutrophil-driven inflammation through cytokine release to facilitate axon regeneration after spinal cord injury in zebrafish. HIGHLIGHTSO_LITime-resolved single-cell transcriptomics of zebrafish spinal cord regeneration. C_LIO_LISpinal cord injury induces fibroblast dedifferentiation. C_LIO_LIDedifferentiated fibroblasts sequentially induce and resolve inflammation. C_LIO_LIDysregulation of inflammation dynamics alters mechano-structural tissue properties. C_LI

neuroscience↗

bioRxiv2025

The 2.3 A Structure of A21, a Protein Component of the Conserved Poxvirus Entry-Fusion Complex

Poxviruses are exceptional in having an entry-fusion complex (EFC) consisting of eleven conserved proteins embedded in the membrane of mature virions. With the goal of understanding the function of the EFC, extensive efforts have been made to determine the structures and roles of its components, and to date, structures have been determined for nine of the eleven proteins. Here, we report the crystal structure of A21, the 10th EFC protein, comprising two -helices clasping a twisted antiparallel {beta}-sheet stabilized by two conserved disulfide bonds. The stability of each of the three A21 loops is provided by hydrogen bonds between main-chain atoms and several highly conserved residues, making the overall fold of A21 and its orthologs resilient to evolutionary change. Based on AlphaFold modeling and phylogenetic analysis of A21, we suggest that its highly conserved N-terminal transmembrane domain and C-terminal -helix enable A21 integration into EFC, where it primarily interacts with the G3/L5 subcomplex and the smallest of EFC components, the O3 protein. HighlightsO_LIWith the structure of A21 determined by us, the structures of 10 out of the 11 proteins forming the entry-fusion complex (EFC) of the poxvirus are now known. C_LIO_LIA21 features a highly conserved transmembrane (TM) domain, which, along with the C--helix, facilitates the integration of A21 and its orthologs into the EFC. C_LIO_LIWithin the EFC, A21 primarily interacts with the G3/L5 subcomplex and the O3 peptide. C_LI

molecular biology↗

bioRxiv2024

CRISPR-Cas9 RALA knockout and reconstitution. Detection and role of RALA S194 phosphorylation in RAS-dependent and RAS-independent cancers.

Downstream of oncogenic RAS, RALA is critical for cancer tumorigenesis, possibly regulated by phosphorylation of its Serine194 residue. We made CRISPR-Cas9 RALA knockout (RALA KO) in three RAS-dependent and two RAS-independent cancer cells. Detection of RALA S194 phosphorylation using the commercial anti-phospho-RALA antibody lacks specificity in all three RAS-dependent cancers. siRNA knockdown of RALA and AURKA inhibition by MLN8237 (VMLN) also did not affect pS194RALA detection in these cancers. RALA KO MiaPaCa2 (RAS-dependent) and MCF7 (RAS-independent) cells, stably reconstituted with WT-RALA and S194A-RALA mutants, showed no effect on RALA activation. Tumour growth was, however, restored partly by WT-RALA, but not S194A-RALA mutant. Thus, RALA S194 phosphorylation is needed for tumor formation, not affecting its activation but possibly through its localization.

cancer biology↗

bioRxiv2024

Unveiling Cell Wall Structure and Echinocandin Response in Candida auris and Candida albicans via Solid-State NMR

Invasive candidiasis affects 1.6 million people annually, implicating high mortality and morbidity in immunocompromised and hospitalized patients. Echinocandins, inhibitors of {beta}-1,3-glucan synthesis, are used as a first-line treatment; however, their efficacy is increasingly compromised by resistance and tolerance. To understand how echinocandins remodel Candida cell wall structures, thereby reducing drug effectiveness, this study compares the effects of echinocandin exposure on the cell walls of the prevalent pathogen Candida albicans and the recently emerged multidrug-resistant superbug Candida auris. High-resolution solid-state NMR analysis revealed a conserved cell wall structure in both species, with a rigid inner layer composed of closely associated chitin microfibrils and {beta}-1,3-glucans, supported by a flexible network of {beta}-1,6-glucans and additional {beta}-1,3-glucans. Despite the presence of N-mannan fibrils in the outer layer, mannan components are mobile and rely on -1,2-linked mannoside sidechains to maintain contact with chitin and {beta}-1,3-glucans. Caspofungin treatment rigidifies certain mannan sidechains and {beta}-1, 6-glucans to reinforce the cell wall in response to the depletion of most {beta}-1,3-glucans. While caspofungin treatment reduced water permeability in both species, only C. albicans responded by inducing cell wall thickening and changes in chitin and {beta}-1,3-glucan dynamics. Furthermore, the deletion of KRE6 genes encoding {beta}-1,6-glucan synthase reduced the echinocandin susceptibility of C. auris, and the impaired {beta}-1,6-glucan biosynthesis were offset by compensatory upregulation of this wall component due to caspofungin treatment. The profound alterations induced by caspofungin in Candida cell wall architecture suggest that cell wall structural contribute substantially to drug resistance and tolerance.

microbiology↗

bioRxiv2024

CDR2 is a dynein adaptor recruited by kinectin to regulate ER sheet organization

The endoplasmic reticulum (ER) relies on the microtubule cytoskeleton for distribution and re-modelling of its extended membrane network, but how microtubule-based motors contribute to ER organization remains unclear. Using biochemical and cell-based assays, we identify cerebellar degeneration-related protein 2 (CDR2) and its paralog CDR2-like (CDR2L), onconeural antigens with poorly understood functions, as ER adaptors for cytoplasmic dynein-1 (dynein). We demonstrate that CDR2 is recruited by the integral ER membrane protein kinectin (KTN1) and that double knockout of CDR2 and CDR2L enhances KTN1-dependent ER sheet stacking, reversal of which by exogenous CDR2 requires its dynein-binding CC1 box motif. Exogenous CDR2 expression additionally promotes CC1 box-dependent clustering of ER sheets near centrosomes. CDR2 competes with the eEF1B{beta} subunit of translation elongation factor 1 for binding to KTN1, and eEF1B{beta} knockdown increases endogenous CDR2 levels on ER sheets, inducing their centrosome-proximal clustering. Our study describes a novel molecular pathway that implicates dynein in ER sheet organization and may be involved in the pathogenesis of paraneoplastic cerebellar degeneration.

cell biology↗

bioRxiv2024

Local Mean Gradient Pattern (LMGP): A novel approach made for the classification of brain CT scan images

ObjectiveThe visual descriptor methods like Local Binary Pattern (LBP) capture anatomical structures in captured images along with their disparities, which can be exploited by suitable methods for the diagnosis of medical anomalies. We developed a Local Mean Gradient Pattern (LMGP), based partly on LBP, a feature extraction algorithm for the classification of Computed Tomography (CT) images of the brain into normal, ischemic, or hemorrhage categories. MethodsThe ASID dataset comprises of 397 acute ischemic stroke CT scan images and the Kaggle dataset consists of 200 CT scan images, out of which, 100 images were of the abnormal brain type (hemorrhage), and the remaining 100 images which dont show any abnormalities were categorized as normal cases. The results of LMGP were compared with eight other feature extraction methods using linear, sigmoid, polynomial, and RBF kernels of the SVM classifier. ResultsThe effectiveness of our methodology was evaluated using recall, logarithmic loss, accuracy (ACC), confusion matrix and area under curve (ROC) metrics. The LMGP performed best when RBF-SVM was used for the classification and gave an accuracy of 93% and 95% in the case of five-fold and ten-fold cross-validation respectively. The ten-fold cross-validation gave a precision score of 0.9410, a recall score of 0.9521, an F1 score of 0.9458, and a negative log loss of -0.1645. ConclusionLMGP combines a unique and robust approach of CT scan image feature extraction by combining local information along with gradient change in pixels of the image. The present results of the study suggest the improved performance of the LMGP method over other methods compared in this study effectively.

bioengineering↗

bioRxiv2024

Machine learning, molecular docking and simulation studies reveal Lomitapide, Lodipamide, Zafirlukast, Netupitant and Salmon Calcitonin as potent drug molecules against Chagas disease

Trypanosoma cruzi (T. cruzi), a protozoan parasite, is the pathogen that causes the hazardous disease Chagas, sometimes referred to as American trypanosomiasis. Currently, there are just two drugs commercially available to treat this fatal condition. So, there is an urgent requirement to create novel, safer and efficient anti-Tc drugs. In this study, we introduce a robust experimental strategy that integrates machine learning with molecular docking and simulation studies to identify the most potential drug candidates from the DrugBank dataset for treating the Chagas disease. In a machine learning method, different classifiers (Naive Bayes, Random Forest, SMO, and C4.5) were used to train the model on the PubChem dataset and the most effective model (C4.5 algorithm) was then chosen and tested on the DrugBank dataset (containing FDA-approved and investigational drugs). The C4.5 algorithm-based machine learning model with an accuracy of 65% predicts the possible drug candidates (a total of 280 drugs were predicted). AutoDock4.2 software was then used to dock the predicted compounds that had a confidence interval of 80% and above. As a result, 47 predicted drugs were docked, and the best of the 5 drugs based on their docking score were chosen for performing the MD simulation studies. MD simulations (100 ns) were conducted for each of the protein-ligand complexes, which produces an average RMSD score (2.23 [A]). The RMSF value of the cruzain (Cys25) binding pocket, after interaction with the five compounds were found to be less than 3.0 [A]. Therefore, based on the molecular docking and simulation studies, we found out that Lomitapide, Lodipamide, Zafirlukast, Netupitant and Salmon Calcitonin have shown a stronger binding affinity with the crystal structure of cruzain (Cyst25) molecule, making them potential effective inhibitors. Hence, we could anticipate that our five computationally validated drugs will soon be available at the clinical trial stage and eventually be made accessible to the necessary Chagas disease patients.

bioinformatics↗

bioRxiv2024

Biallelic Loss of Molecular Chaperone Molecule AIP Results in a Novel Severe Multisystem Disease Defined by Defective Proteostasis

Children born with deleterious biallelic variants of the chaperone aryl hydrocarbon receptor interacting protein (AIP) have a novel pediatric metabolic disease presenting a severe, complex clinical phenotype characterized by failure to develop following birth. Analysis of Aip knockout mouse embryonic fibroblasts and patient-derived dermal fibroblasts revealed that AIP was required to support proteostasis; including proteasome activity, induction of autophagy and lysosome function. aip knockout zebrafish, recapitulated the phenotype of the children; dying at an early stage of development when autophagy is required to adapt to periods of starvation. Our results demonstrate that AIP plays a crucial role in initiating autophagy and maintaining proteostasis in vitro and in vivo. One Sentence SummaryHomozygous loss of the chaperone AIP results in a novel pediatric disease exhibiting multiple features of a lysosomal storage disease.

molecular biology↗

bioRxiv2024

Insulin signaling accelerates the anterograde movement of Rab4 vesicles in axons through Klp98a/KIF16B recruitment via Vps34-PI3Kinase

Rab4 GTPase organizes endosomal sorting essential for maintaining the balance between recycling and degradative pathways. Rab4 localizes to many cargos whose transport in neurons is critical for regulating neurotransmission and neuronal health. Furthermore, elevated Rab4 levels in the CNS are associated with synaptic atrophy and neurodegeneration in Drosophila and humans, respectively. However, how the transport of Rab4-associated vesicles is regulated in neurons remains unknown. Using in vivo time-lapse imaging of Drosophila larvae, we show that activation of insulin signaling via Dilp2 and dInR increases the anterograde velocity, run length, and flux of Rab4 vesicles in the axons. Molecularly, we show that activation of neuronal insulin signaling further activates Vps34, elevates the levels of PI(3)P on Rab4-associated vesicles, recruits Klp98A (a PI(3)P-binding kinesin-3 motor) and activates their anterograde transport. Together, these observations delineate the role of insulin signaling in regulating axonal transport and synaptic homeostasis. HighlightsO_LIDilp2-mediated insulin signaling activates anterograde transport of Rab4 vesicles C_LIO_LIVps34 regulates anterograde velocity, run length and flux of Rab4 vesicles C_LIO_LILocal PI(3)P signaling on Rab4 vesicles regulates their motility in the axons C_LIO_LIPI(3)P production upon acute insulin stimulation recruits Klp98A on Rab4 vesicles C_LI

cell biology↗

bioRxiv2024

Cell-specific polymerization-driven biomolecular condensate formation fine-tunes root tissue morphogenesis

Formation of biomolecular condensates can be driven by weak multivalent interactions and emergent polymerization. However, the mechanism of polymerization-mediated condensate formation is less studied. We found lateral root cap cell (LRC)-specific SUPPRESSOR OF RPS4-RLD1 (SRFR1) condensates fine-tune primary root development. Polymerization of the SRFR1 N-terminal domain is required for both LRC condensate formation and optimal root growth. Surprisingly, the first intrinsically disordered region (IDR1) of SRFR1 can be functionally substituted by a specific group of intrinsically disordered proteins known as dehydrins. This finding facilitated the identification of functional segments in the IDR1 of SRFR1, a generalizable strategy to decode unknown IDRs. With this functional information we further improved root growth by modifying the SRFR1 condensation module, providing a strategy to improve plant growth and resilience.

molecular biology↗

bioRxiv2024

IL-7 mediated upregulation of VLA-4 increases accumulation of adoptively transferred T lymphocytes in murine glioma.

The efficacy of T cell-activating therapies against glioma is limited by an immunosuppressive tumor microenvironment and tumor-induced T cell sequestration. We investigated whether peripherally infused non-antigen specific autologous lymphocytes (ALT) could accumulate in intracranial tumors. We observed that non-specific autologous CD8+ ALT cells can indeed accumulate in this context, despite endogenous T cell sequestration in bone marrow. Rates of intratumoral accumulation were markedly increased when expanding lymphocytes with IL-7 compared to IL-2. Pre-treatment with IL-7 ALT also enhanced the efficacy of multiple tumor-specific and non-tumor-specific T cell-dependent immunotherapies against orthotopic murine and human xenograft gliomas. Mechanistically, we detected increased VLA-4 on mouse and human CD8+ T cells following IL-7 expansion, with increased transcription of genes associated with migratory integrin expression (CD9). We also observed that IL-7 increases S1PR1 transcription in human CD8+ T cells, which we have shown to be protective against tumor-induced T cell sequestration. These observations demonstrate that expansion with IL-7 enhances the capacity of ALT to accumulate within intracranial tumors, and that pre-treatment with IL-7 ALT can boost the efficacy of subsequent T cell-activating therapies against glioma. Our findings will inform the development of future clinical trials where ALT pre-treatment can be combined with T cell-activating therapies. Brief SummaryT cell immunotherapies are limited by few T cells in glioma. Adoptively transferred lymphocytes expanded with IL-7 exhibit increased VLA-4 expression and accumulate in tumors.

cancer biology↗

bioRxiv2024

Bridging fungal resistance and plant growth through constitutive overexpression of Thchit42 gene in Pelargonium graveolens

Pelargonium graveolens essential oil possesses significant attributes, known for perfumery and aromatherapy. However, optimal yield and propagation are predominantly hindered by biotic stress. All biotechnological approaches have yet to prove effective in addressing fungal resistance. The current study developed transgenic geranium bridging molecular mechanism of fungal resistance and plant growth by introducing cassette 35S::Thchit42. Furthermore, 120 independently putative transformed explants were regenerated on kanamycin fortified medium. Primarily transgenic lines were demonstrated peak pathogenicity and antifungal activity against formidable Colletotrichum gloeosporioides and Fusarium oxysporum. Additionally, phenotypic analysis revealed [~]2fold increase in leaf size and [~]2.1fold enhanced oil content. To elucidate the molecular mechanisms for genotypic cause, De novo transcriptional profiles were analyzed to indicate that the auxin-regulated longifolia gene is accountable for augmentation in leaf size, and ZF RICESLEEPER attributes growth upregulation. Collectively, data provides valuable insights into unravelling the mechanism of Thchit42-mediated crosstalk between morphological and chemical alteration in transgenic plants. This knowledge might create novel opportunities to cultivate fungal-resistant geranium throughout all seasons to fulfil demand.

plant biology↗

bioRxiv2024

Cross-species modeling and enhancement of cognitive control with striatal brain stimulation

Brain disorders, particularly mental disorders, might be effectively treated by direct electrical brain stimulation, but clinical progress requires understanding of therapeutic mechanisms. Animal models have not helped, because there are no direct animal models of mental illness. We show a path past this roadblock, by leveraging a common ingredient of most mental disorders: impaired cognitive control. We previously showed that deep brain stimulation (DBS) improves cognitive control in humans. We now reverse translate that result, showing that DBS-like stimulation of the mid-striatum improves cognitive control in rats. Using this model, we identify a mechanism, improvement in domain-general cognitive control, and rule out competing hypotheses such as impulsivity. The rat findings explain prior human results and have immediate implications for clinical practice and future trial design. One Sentence Summary: Developing a reliable animal model of a human brain stimulation therapy reveals that this therapy works by enhancing the brains ability to process conflicting pieces of evidence.

neuroscience↗

bioRxiv2024

Neuronal microstructural changes in the human brain are associated with neurocognitive aging

Gray matter (GM) alterations play a role in aging-related disorders like Alzheimers disease and related dementias, yet MRI studies mainly focus on macroscopic changes. Although reliable indicators of atrophy, morphological metrics like cortical thickness lack the sensitivity to detect early changes preceding visible atrophy. Our study aimed at exploring the potential of diffusion MRI in unveiling sensitive markers of cortical and subcortical age-related microstructural changes and assessing their associations with cognitive and behavioral deficits. We leveraged the Human Connectome Project-Aging cohort that included 707 unimpaired participants (394 female; median age = 58, range = 36-90 years) and applied the powerful mean apparent diffusion propagator model to measure microstructural parameters, along with comprehensive behavioral and cognitive test scores. Both macro- and microstructural GM characteristics were strongly associated with age, with widespread significant microstructural correlations reflective of cellular morphological changes, reduced cellular density, increased extracellular volume, and increased membrane permeability. Importantly, when correlating MRI and cognitive test scores, our findings revealed no link between macrostructural volumetric changes and neurobehavioral performance. However, we found that cellular and extracellular alterations in cortical and subcortical GM regions were associated with neurobehavioral performance. Based on these findings, it is hypothesized that increased microstructural heterogeneity and decreased neurite orientation dispersion precede macrostructural changes, and that they play an important role in subsequent cognitive decline. These alterations are suggested to be early markers of neurocognitive performance that may distinctly aid in identifying the mechanisms underlying phenotypic aging and subsequent age-related functional decline.

neuroscience↗

bioRxiv2023

Impact of CRISPR/HDR-editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) requires both sufficient HDR efficiency and protection of LT-HSC function and number. The impact of HDR on true LT-HSCs clonal dynamics in a relevant large animal model has not previously been studied. To track the HDR-edited cells, autologous rhesus macaque (RM) CD34+ cells were electroporated with the gRNA/Cas9 ribonucleoprotein (RNP) and HDR cassette barcode library structure and reinfused into RMs following myeloablation. For competitive model animals, fractionated CD34+ cells were transduced with a barcoded GFP-expressing lentiviral vector (LV) and electroporated via HDR machinery, respectively. CD33 knockout (KO) neutrophils were prevalent early following engraftment and then rapidly decreased, resulting in less than 1% total editing efficiency. Interestingly, in competitive animals, a higher concentration of i53 mRNA result in a less steep reduction in CD33 KO cells, presented a modest decrease in HDR rate (0.1-0.2%) and total indels (1.5-6.5%). In contrast, the drop off of LV-transduced GFP+ cells stabilized at 20% after 2 months. We next retrieved embedded barcodes and revealed that various clones contributed to early hematopoietic reconstitution, then after dominant clones appeared at steady state throughout the animals. In conclusion, CRISPR/HDR edited cells disappeared rapidly after the autologous transplantation in RM despite substantial gene editing outcome, whereas LV-transduced cells were relatively well maintained. Clonality of HDR-edited cells drastically shrank at early stage and then relied on several dominant clones, which can be mildly mitigated by the introduction of i53 mRNA.

cell biology↗