Biology subjects

Nelson, J.

Publications and source records attributed to Nelson, J..

At least 19 recordsLinked to original sources

bioRxiv2026

LNP Delivery of Prime Editors Enables Durable Correction of PAH R408W in Phenylketonuria Models

Phenylketonuria (PKU) is a monogenic metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene, the most common of which is c.1222C>T (p.R408W). Current treatment options, including dietary restriction and enzyme substitution, can be effective but are often challenging for patients to maintain and do not address the underlying genetic defect. Gene editing represents a compelling one-time treatment approach for PKU. Herein, we demonstrate mRNA:LNP-mediated delivery of prime editors that enable therapeutically-relevant levels of correction of the R408W mutation in both in vitro and in vivo model systems. Further, we found that co-delivery of the DNA repair modulator RAD18 and engineering of the prime editor protein, enabled 2-fold and 3-fold increases in prime editing relative to PE3 respectively, resulting in ~50% correction efficiency in a humanized PKU mouse model. Collectively, this work identifies novel potential approaches to enhancing prime editing and establishes LNP-delivered prime editing as a mutation-specific, non-viral therapeutic strategy for PKU, as well as providing a framework for extending prime editing to other inherited liver diseases.

pharmacology and toxicology↗

bioRxiv2026

Annotation-Based Gene-Peak Links Improve Regulatory Network Prediction of Gene Expression in Human Kidney Multi-Omics

BackgroundLinking distal regulatory elements to their target genes is a central problem for interpreting chromatin accessibility and other non-coding genomic data. Proximity-based mapping is convenient but ignores three-dimensional enhancer-promoter architecture and can misassign long-range regulatory effects. Correlation-based approaches can also miss regulatory links because of limited statistical power and restrictive distance or significance thresholds. Single-cell and single-nucleus multi-omic datasets, such as 10x Multiome profiles that jointly measure chromatin accessibility and gene expression in the same cells or nuclei, now provide a way to evaluate gene-peak linkage strategies by testing how well linked accessibility features predict gene expression. Existing methods often focus on scoring individual enhancer-gene pairs. In this study, we proposed and constructed a fast annotation-based candidate gene-peak network and tested whether it improves downstream prediction of gene expression. MethodsWe first built a unified gene-peak regulatory network by integrating enhancer-based, promoter-based, and proximity-based linkage strategies. We then used single-cell multiome data from the Kidney Precision Medicine Project (KPMP) 10x Multiome cohort to evaluate whether the proposed links captured regulatory signals. We aggregated RNA expression and ATAC accessibility at the cell-type cluster level and trained predictive models to evaluate how well different linkage strategies could explain gene expression based on accessibility. Model performance was compared between annotation-based (including both enhancer- and promoter-based links) and proximity-based gene-peak links using testing R{superscript 2} and mean squared error (MSE) in a strict set of 1,704 genes and an adaptive set of 7,973 genes with more relaxed requirements. ResultsIn the strict regime (1,704 genes with [≥]20 peaks assigned by the nearest-TSS rule and [≥]10 annotated enhancer-based peaks), the annotation-based model consistently achieved higher testing R{superscript 2} and lower testing MSE than the proximity-based model. In the larger adaptive regime (7,973 genes with [≥]5 proximity-based and [≥]2 enhancer-based peaks), we defined for each gene a balanced number of selected peaks based on its available closest and enhancer links; the annotation-based model again showed globally higher testing R{superscript 2} and lower testing MSE. These improvements were observed over a broad range of candidate-linked peak numbers. ConclusionsUsing human kidney 10x Multiome data, we show that a fast annotation-based gene-peak linkage framework can improve prediction of gene expression from chromatin accessibility compared with conventional approaches. These results support the use of biologically informed enhancer and promoter annotations when constructing candidate gene regulatory networks. Our framework also showed concordance with the correlation-based Signac LinkPeaks method while providing broader coverage and greater computational efficiency. We have implemented these annotation-based linkage methods in the GPlinksR R package, providing a fast and scalable tool for constructing regulatory networks.

genomics↗

bioRxiv2026

Comparison of Galleria mellonella, Epithelial Cell Cytotoxicity, and Mouse Model of Bacteremia to Measure Pseudomonas aeruginosa Virulence

Considerable effort has focused on identifying alternatives to mouse models in research studies. In the field of bacterial pathogenesis, Galleria mellonella and epithelial cell lines have been widely used for this purpose, but the concordance of these models with mice remains unclear. To begin to address this knowledge gap, we used 105 clinical isolates of Pseudomonas aeruginosa for which the virulence had been previously determined in a mouse bacteremia model. A semistrong correlation was observed between G. mellonella median time to 50% mortality and mouse 50% pre-lethal dose (LD50) values (Spearmans rank correlation coefficient [{rho}] = 0.75), whereas percent A549 epithelial-like cell lysis during co-culture showed a weak correlation to mouse LD50 values ({rho} = -0.47). Given the stronger correlation between G. mellonella and mouse virulence, we next examined whether G. mellonella could substitute for mice when asking questions about the virulence of large numbers of P. aeruginosa isolates. Results from mice indicated that isolates with resistance to more antibiotics were significantly less virulent, and the use of G. mellonella identified the same inverse correlation. Furthermore, both models found no evidence for the existence of hypervirulent clonal lineages. In particular, isolates belonging to sequence types defined as high-risk clones were not consistently more virulent than other isolates, despite the known association of high-risk clones with poor clinical outcomes. These findings suggest that G. mellonella can serve as an adequate substitute for mice when addressing specific population-based virulence questions, although conclusions should be confirmed in mice. Author SummaryWe found that virulence measurements in a G. mellonella infection model showed a semistrong correlation with those from a mouse bacteremia model and that this insect larval model adequately detected population-level trends similarly to mice. In contrast, A549 epithelial-like cell lysis during bacterial co-culture correlated less well with mouse virulence. Together, these results support the use of G. mellonella as a scalable, low-cost, and humane first-line model for assessing P. aeruginosa virulence but also indicate that conclusions should be validated in mice.

microbiology↗

bioRxiv2025

The G Protein-Coupled Receptor GPR31 Promotes Pro-inflammatory Responses in Pancreatic Islets and Macrophages

In type 1 diabetes (T1D), the innate and adaptive immune systems attack and eventually destroy the insulin-secreting pancreatic {beta} cells. During this process, {beta} cells activate inflammatory signaling pathways that augment the dysfunction and destruction imposed by cellular autoimmunity. The 12-lipoxygenase (12-LOX) pathway produces the pro-inflammatory eicosanoid 12-HETE, which induces oxidative and endoplasmic reticulum stress and results in diminished insulin secretion and apoptosis. The G protein-coupled receptor GPR31 has been identified as a putative receptor for 12-HETE. In this study, we generated conventional GPR31 knockout (KO) mice on the C57BL/6J background. To interrogate the role of GPR31 in {beta} cells, we treated islets from wildtype and Gpr31b KO mice with pro-inflammatory cytokines and subjected the islets to RNA sequencing. Differentially expressed pathways in Gpr31b KO islets included those pertaining to inflammation and oxidative stress, consistent with functional studies that demonstrated reduced cytokine-induced oxidative stress in Gpr31b KO islets compared to wildtype controls. Bone marrow-derived macrophages from Gpr31b KO mice showed reduced macrophage migration and decreased inflammatory IFN- and IFN-{gamma} signaling by RNA sequencing. To mimic islet and macrophage inflammation as seen in T1D, wildtype and Gpr31b KO mice were treated with the diabetogenic toxin streptozotocin. Compared to wildtype, Gpr31b KO mice had improved glucose tolerance and preserved {beta}-cell mass. siRNA knockdown of Gpr31b in non-obese diabetic (NOD) mice reduced insulitis, macrophage infiltration, and oxidative stress. Collectively, these findings are consistent with previously published data using 12/15-LOX KO mice and suggest that GPR31 mediates the pro-inflammatory responses of 12-HETE in both {beta} cells and macrophages.

molecular biology↗

bioRxiv2025

A New Type of Nonsuppressible Viremia Produced by HIV-Infected Macrophage

BackgroundHIV-1 RNA typically declines rapidly after initiation of antiretroviral therapy (ART); often reaching undetectable levels within a few weeks and remaining undetectable by standard assays. However, some patients on ART have persistent nonsuppressible viremia (NSV) that does not respond to treatment optimization or intensification. NSV can emerge at the time of ART initiation (primary NSV) or after being ART-suppressed (secondary NSV). Here, we examine mechanisms producing primary NSV in four people on ART. MethodsBlood samples were collected from four participants who, despite being adherent to ART, required approximately a year or more to become virologically suppressed. Viral RNA and proviral DNA genomes were sequenced to examine HIV-1 drug resistance, genome intactness and genetic diversity. The ability of HIV-1 Envs to facilitate efficient entry into cells expressing low levels of CD4 (a proxy for macrophage tropism) was assessed. ResultsBefore ART, the blood contained HIV-1 RNA genomes that were adapted to replication in CD4+ T cells and rapidly decayed after ART initiation. During ART, the blood contained HIV-1 genomes that were drug sensitive, genetically diverse, macrophage-tropic, not evolving and often had defects in vpr. ConclusionsOur results suggest that in individuals with primary NSV, ART stopped virus replication, but large pools of long-lived, HIV-infected macrophage continued to produce virus. This is mechanistically distinct from secondary NSV produced by CD4+ T cell clones. In addition, defects in vpr independently accumulation in macrophage-tropic lineages found in three participants, suggesting that vpr may impact survival of, or virus production from, HIV-infected macrophage. SUMMARYPeople with HIV-1 on ART can experience persistent, nonsuppressible viremia (NSV). We identified four people with a previously uncharacterized form of NSV that emerged at the time of ART initiation (primary NSV) and is notable for its genetic diversity, drug sensitivity, and macrophage tropism. This is mechanistically distinct from previously described cases of NSV produced by CD4+ T cells in people who were previously ART-suppressed (secondary NSV).

microbiology↗

bioRxiv2025

Distinct cell types along thick ascending limb express pathways for monovalent and divalent cation transport

Kidney thick ascending limb cells reabsorb sodium, potassium, calcium, and magnesium and contribute to urinary concentration. These cells are typically viewed as of a single type that recycles potassium across the apical membrane and generates a lumen-positive transepithelial voltage driving calcium and magnesium reabsorption, although variability in potassium channel expression has been reported. Additionally, recent transcriptomic analyses suggest that different cell types exist along this segment, but classifications have varied and have not led to a new consensus model. We used immunolocalization, electrophysiology and enriched single nucleus RNA-Seq to identify thick ascending limb cell types in rat, mouse and human. We identified three major TAL cell types defined by expression of potassium channels and claudins. One has apical potassium channels, low basolateral potassium conductance, and is bordered by a sodium-permeable claudin. A second lacks apical potassium channels, has high basolateral potassium conductance and is bordered by calcium- and magnesium-permeable claudins. A third type also lacks apical potassium channels and has a high basolateral potassium conductance, but these cells are ringed by sodium-permeable claudins. The recognition of diverse cell types resolves longstanding questions about how solute transport can be modulated selectively and how disruption of these cells leads to human disease.

physiology↗

bioRxiv2024

Horizontal gene transfers and terpene metabolism drive plant-fungal interaction in Marchantia polymorpha

The liverwort Marchantia polymorpha has emerged as a model for studying plant immunity in bryophytes, providing unique insights into conserved defense mechanisms across land plants. By contrast, Marchantia-specific immune mechanisms have not been explored. In this study, we investigated the genetic basis of quantitative resistance in M. polymorpha against the fungal pathogen Colletotrichum nymphaeae, a naturally occurring compatible parasite. Through a combination of phenotypic, cytological and transcriptomic approaches, combined with genome-wide association studies (GWAS), we identified key defense-related genes and pathways. Transcriptomic analyses performed on two lines with contrasting suceptibilities to the pathogen revealed a strong overlap in immune responses between M. polymorpha and angiosperms, including the upregulation of PR proteins, transcription factors typically associated with biotic stresses, and enzymes involved in specialized metabolism. Leveraging the biological and genetic variability present in a collection of natural M. polymorpha accessions, highlight the role of horizontally transferred microbial-like terpene synthase (MTPSL) genes, which may contribute to the exceptional terpene diversity in liverworts and play a role in pathogen resistance. GWAS uncovered candidate loci associated with resistance traits, implicating both core immune components and specialized metabolic pathways. These results provide new insights into the specific molecular underpinnings of bryophyte immunity and underscore the evolutionary significance of horizontal gene transfer and specialized metabolites in shaping plant-pathogen interactions.

plant biology↗

bioRxiv2024

Locating the Missing Chlorophylls f in Far-red Photosystem I

The discovery of chlorophyll f-containing oxygenic photosynthesis, with its long-wavelength photochemistry, represented a new low-energy paradigm. However, subsequent structural studies on chlorophyll f-containing Photosystem I (PSI) found five chlorophylls f but none among the photochemically active pigments and concluded that chlorophyll f plays no photochemical role. Here we report a cryo-EM structure (2.01 [A]) of far-red PSI from Chroococcidiopsis thermalis PCC 7203, showing all eight chlorophylls f, including the redox active A-1B. Simulations of absorption difference spectra induced by charge separation indicate that the A-1B chlorophyll f absorbs at 755 nm. The chlorophyll f sites, some wavelength assignments, and conserved far-red-specific amino acids, provide functional insights, including redox tuning of chlorophyll f as the primary donor and far-red excitation energy-sharing over the PSI trimer.

biochemistry↗

bioRxiv2024

Single-cell and spatiotemporal profile of ovulation in the mouse ovary

Ovulation is a spatiotemporally coordinated process that involves several tightly controlled events, including oocyte meiotic maturation, cumulus expansion, follicle wall rupture and repair, and ovarian stroma remodeling. To date, no studies have detailed the precise window of ovulation at single-cell resolution. Here, we performed parallel single-cell RNA-seq and spatial transcriptomics on paired mouse ovaries across an ovulation time course to map the spatiotemporal profile of ovarian cell types. We show that major ovarian cell types exhibit time-dependent transcriptional states enriched for distinct functions and have specific localization profiles within the ovary. We also identified gene markers for ovulation-dependent cell states and validated these using orthogonal methods. Finally, we performed cell-cell interaction analyses to identify ligand-receptor pairs that may drive ovulation, revealing previously unappreciated interactions. Taken together, our data provides a rich and comprehensive resource of murine ovulation that can be mined for discovery by the scientific community.

bioinformatics↗

bioRxiv2024

DERIVATION OF ELEPHANT INDUCED PLURIPOTENT STEM CELLS

The crisis of biodiversity loss in the anthropogenic era requires new tools for studying non-model organisms. Elephants, for example, are both an endangered species and excellent models studying complex phenotypes like size, social behavior, and longevity, but they remain severely understudied. Here we report the first derivation of elephant (Elephas maximus) induced pluripotent stem cells (emiPSCs) achieved via a two-step process of chemical-media induction and colony selection, followed by overexpression of elephant transcription factors OCT4, SOX2, KLF4, MYC {+/-} NANOG and LIN28A, and modulation of the TP53 pathway. Since the seminal discovery of reprogramming by Shinya Yamanaka, iPSCs from many species including the functionally extinct northern white rhinocerous have been reported, but emiPSCs have remained elusive. While for multiple species the reprogramming protocol was adopted with little changes compared to model organisms like mouse and human, our emiPSC protocol requires a longer timeline and inhibition of TP53 expansion genes that are hypothesized to confer unique cancer resistance in elephants. iPSCs unlock tremendous potential to explore cell fate determination, cell and tissue development, cell therapies, drug screening, disease modeling, cancer development, gametogenesis and beyond to further our understanding of this iconic megafauna. This study opens new frontiers in advanced non-model organism cellular models for genetic rescue and conservation.

cell biology↗

bioRxiv2024

Mapping in silico genetic networks of the KMT2D tumour suppressor gene to uncover novel functional associations and cancer cell vulnerabilities

Loss-of-function (LOF) alterations in tumour suppressor genes cannot be directly targeted. Approaches characterising gene function and vulnerabilities conferred by such mutations are required. Here, we computationally map genetic networks of KMT2D, a tumour suppressor gene frequently mutated in several cancer types. Using KMT2D loss-of-function (KMT2DLOF) mutations as a model, we illustrate the utility of in silico genetic networks in uncovering novel functional associations and vulnerabilities in cancer cells with LOF alterations affecting tumour suppressor genes. We revealed genetic interactors with functions in histone modification, metabolism, and immune response, and synthetic lethal (SL) candidates, including some encoding existing therapeutic targets. Analysing patient data from The Cancer Genome Atlas and the Personalized OncoGenomics Project, we showed, for example, elevated immune checkpoint response markers in KMT2DLOF cases, possibly supporting KMT2DLOF as an immune checkpoint inhibitor biomarker. Our study illustrates how tumour suppressor gene LOF alterations can be exploited to reveal potentially targetable cancer cell vulnerabilities.

bioinformatics↗

bioRxiv2023

Systematic benchmarking of imaging spatial transcriptomics platforms in FFPE tissues

Emerging imaging spatial transcriptomics (iST) platforms and coupled analytical methods can recover cell-to-cell interactions, groups of spatially covarying genes, and gene signatures associated with pathological features, and are thus particularly well-suited for applications in formalin fixed paraffin embedded (FFPE) tissues. Here, we benchmarked the performance of three commercial iST platforms on serial sections from tissue microarrays (TMAs) containing 23 tumor and normal tissue types for both relative technical and biological performance. On matched genes, we found that 10x Xenium shows higher transcript counts per gene without sacrificing specificity, but that all three platforms concord to orthogonal RNA-seq datasets and can perform spatially resolved cell typing, albeit with different false discovery rates, cell segmentation error frequencies, and with varying degrees of sub-clustering for downstream biological analyses. Taken together, our analyses provide a comprehensive benchmark to guide the choice of iST method as researchers design studies with precious samples in this rapidly evolving field.

cancer biology↗

bioRxiv2023

The Marchantia pangenome reveals ancient mechanisms of plant adaptation to the environment

Plant adaptation to a terrestrial life 450 million years ago played a major role in the evolution of life on Earth. This shift from an aquatic environment has been mostly studied by focusing on flowering plants. Here, we gathered a collection of 133 accessions of the non-vascular plants Marchantia polymorpha and studied its intraspecific diversity using selection signature analyses, genome-environment association study and a gene-centered pangenome. We identified adaptive features shared with flowering plants, such as peroxidases or nucleotide-binding and leucine-rich repeat (NLR), which likely played a role in the adaptation of the first land plants to the terrestrial habitat. The M. polymorpha pangenome also harbored lineage-specific accessory genes absent from seed plants. We conclude that different land plants lineages still share many elements from the genetic toolkit evolved by their most recent common ancestor to adapt to the terrestrial habitat, refined by lineage specific polymorphisms and gene family evolutions.

plant biology↗

bioRxiv2023

Remyelination protects neurons from DLK-mediated neurodegeneration

Chronic demyelination and oligodendrocyte loss deprive neurons of crucial support. It is the degeneration of neurons and their connections that drives progressive disability in demyelinating disease. However, whether chronic demyelination triggers neurodegeneration and how it may do so remain unclear. We characterize two genetic mouse models of inducible demyelination, one distinguished by effective remyelination and the other by remyelination failure and chronic demyelination. While both demyelinating lines feature axonal damage, mice with blocked remyelination have elevated neuronal apoptosis and altered microglial inflammation, whereas mice with efficient remyelination do not feature neuronal apoptosis and have improved functional recovery. Remyelination incapable mice show increased activation of kinases downstream of dual leucine zipper kinase (DLK) and phosphorylation of c-Jun in neuronal nuclei. Pharmacological inhibition or genetic disruption of DLK block c-Jun phosphorylation and the apoptosis of demyelinated neurons. Together, we demonstrate that remyelination is associated with neuroprotection and identify DLK inhibition as protective strategy for chronically demyelinated neurons. HighlightsO_LICharacterization of a transgenic mouse model of demyelination without subsequent remyelination C_LIO_LIRemyelination protects neurons from axon loss and neuronal apoptosis C_LIO_LIMAPK and c-Jun phosphorylation are increased in mice featuring remyelination failure C_LIO_LIDLK is necessary for the apoptosis of chronically demyelinated neurons C_LI

neuroscience↗

bioRxiv2023

Comparative pathogenesis of two lineages of Powassan virus reveals differing routes of neuroinvasion leading to distinct clinical outcome, neuropathology and inflammation

Tick-borne flaviviruses (TBFV) can cause severe neuroinvasive disease which may result in death or long-term neurological deficit in over 50% of survivors. Multiple mechanisms for invasion of the central nervous system (CNS) by flaviviruses have been proposed including axonal transport, transcytosis, endothelial infection, and Trojan horse routes. Flaviviruses may utilize different or multiple mechanisms of neuroinvasion depending on the specific virus, infection site, and host variability. In this work we have shown that infection of BALB/cJ mice with either Powassan virus lineage I (Powassan virus) or lineage II (deer tick virus) results in distinct spatial tropism of infection in the CNS which correlated with unique clinical presentation for each lineage. Comparative transcriptomics of infected brains demonstrates activation of different immune pathways and downstream host responses. Ultimately the comparative pathology and transcriptomics are congruent with different clinical signs in a murine model. These results suggest that different disease presentations would be occur in clinical cases due to the innate differences in the two lineages of Powassan virus. Author SummaryPowassan virus causes a nationally notifiable disease which can cause severe neurological disease in humans and has no approved vaccines or therapeutics. Although two distinct lineages circulate in North America, clinical differentiation is not typically performed, and pathology has been assumed to be similar between lineages. In this work, a direct comparison of lineage I (Powassan virus) and lineage II (deer tick virus) demonstrated distinct differences in the clinical presentation, pathology of the central nervous system, and immune response in immunocompetent mice. These differences suggest that deer tick virus and Powassan virus do not utilize the same mechanisms for neuroinvasion and dissemination within the CNS. This is clinically relevant as the development of treatment plans and therapeutics need to be evaluated for these virus lineages.

microbiology↗

bioRxiv2023

The ENCODE Uniform Analysis Pipelines

The Encyclopedia of DNA elements (ENCODE) project is a collaborative effort to create a comprehensive catalog of functional elements in the human genome. The current database comprises more than 19000 functional genomics experiments across more than 1000 cell lines and tissues using a wide array of experimental techniques to study the chromatin structure, regulatory and transcriptional landscape of the Homo sapiens and Mus musculus genomes. All experimental data, metadata, and associated computational analyses created by the ENCODE consortium are submitted to the Data Coordination Center (DCC) for validation, tracking, storage, and distribution to community resources and the scientific community. The ENCODE project has engineered and distributed uniform processing pipelines in order to promote data provenance and reproducibility as well as allow interoperability between genomic resources and other consortia. All data files, reference genome versions, software versions, and parameters used by the pipelines are captured and available via the ENCODE Portal. The pipeline code, developed using Docker and Workflow Description Language (WDL; https://openwdl.org/) is publicly available in GitHub, with images available on Dockerhub (https://hub.docker.com), enabling access to a diverse range of biomedical researchers. ENCODE pipelines maintained and used by the DCC can be installed to run on personal computers, local HPC clusters, or in cloud computing environments via Cromwell. Access to the pipelines and data via the cloud allows small labs the ability to use the data or software without access to institutional compute clusters. Standardization of the computational methodologies for analysis and quality control leads to comparable results from different ENCODE collections - a prerequisite for successful integrative analyses. Database URL: https://www.encodeproject.org/

bioinformatics↗

bioRxiv2022

A critical period for neurogenesis loss and cognitive decline in patients with mesial temporal lobe epilepsy patients

Mesial temporal lobe epilepsy (MTLE) is a syndromic disorder presenting with seizures and cognitive comorbidities. While seizure etiology is increasingly understood, the pathophysiological mechanisms contributing to cognitive decline and epilepsy progression remain less recognized. We have previously shown that adult hippocampal neurogenesis, a process contributing to visual spatial learning and memory in rodents, dramatically declines in MTLE patients with increased disease duration. Here, we investigate when multiple cognitive domains become effected by epilepsy disease duration and how human neurogenesis levels contribute to it. We find that intelligence, and verbal learning and memory decline at a critical period of 20 years disease duration. Surprisingly, the number of human immature neurons positively associate with auditory verbal, rather than visuospatial, learning and memory. Moreover, we uncover cognitive functions enriched to either immature or mature granule neurons and functions shared between them. Our study provides cellular evidence of how adult neurogenesis contributes to human cognition, and signifies an opportunity to advance regenerative medicine for patients with MTLE and other cognitive disorders. O_FIG O_LINKSMALLFIG WIDTH=108 HEIGHT=200 SRC="FIGDIR/small/507339v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@12d6c9forg.highwire.dtl.DTLVardef@13670aborg.highwire.dtl.DTLVardef@11efda3org.highwire.dtl.DTLVardef@19de537_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Expanding the scope of bacterial CRISPR activation with PAM-flexible dCas9 variants

CRISPR-Cas transcriptional tools have been widely applied for programmable regulation of complex biological networks. In comparison to eukaryotic systems, bacterial CRISPR activation (CRISPRa) has stringent target site requirements for effective gene activation. While genes may not always have an NGG PAM at the appropriate position, PAM-flexible dCas9 variants can expand the range of targetable sites. Here we systematically evaluate a panel of PAM-flexible dCas9 variants for their ability to activate bacterial genes. We observe that dxCas9-NG provides a high dynamic range of gene activation for sites with NGN PAMs while dSpRY permits modest activity across almost any PAM. Similar trends were observed for heterologous and endogenous promoters. For all variants tested, improved PAM-flexibility comes with the tradeoff that CRISPRi-mediated gene repression becomes less effective. Weaker CRISPRi gene repression can be partially rescued by expressing multiple sgRNAs to target many sites in the gene of interest. Our work provides a framework to choose the most effective dCas9 variant for a given set of gene targets, which will further expand the utility of CRISPRa/i gene regulation in bacterial systems.

synthetic biology↗