Biology subjects

Mohr, S. E.

Publications and source records attributed to Mohr, S. E..

13 recordsLinked to original sources

bioRxiv2026

Genome-wide CRISPR knockout cell screening platform for the disease vector tick species Ixodes scapularis

The black legged tick, Ixodes scapularis, is a vector of the bacterium that causes Lyme disease and several other illnesses, including anaplasmosis, babesiosis, and tick-borne encephalitis. Although high-quality genome annotations are available for I. scapularis, functional understanding of I. scapularis genes is limited. To address this, we developed a platform for genome-wide CRISPR-Cas9 knockout screening in I. scapularis cells. To evaluate the platform, we performed a screen to identify genes associated with cellular fitness, and screens for resistance to treatment with copper chloride, Antimycin A, or Destruxin A (DA), a cyclic hexadepsipeptide produced by the pathogenic fungus Metarhizium anisopliae. In each case, the screens implicate specific sets of conserved and non-conserved I. scapularis genes in relevant cellular functions, providing the first experimental evidence of function for a large set of I. scapularis genes. Altogether, in this first-of-its-kind effort for the arthropod subclass Acari, we present an unbiased genome-wide CRISPR-Cas9 knockout cell screening platform, related resources, and datasets that will be broadly useful to efficiently uncover cellular functions of I. scapularis genes.

genetics↗

bioRxiv2024

A genome-wide CRISPR screen in Anopheles mosquito cells identifies essential genes and required components of clodronate liposome function

Anopheles mosquitoes are the sole vector of human malaria, the most burdensome vector-borne disease worldwide. Strategies aimed at reducing mosquito populations and limiting their ability to transmit disease show the most promise for disease control. Therefore, gaining an improved understanding of mosquito biology, and specifically that of the immune response, can aid efforts to develop new approaches that limit malaria transmission. Here, we use a genome-wide CRISPR screening approach for the first time in mosquito cells to identify essential genes in Anopheles and identify genes for which knockout confers resistance to clodronate liposomes, which have been widely used in mammals and arthropods to ablate immune cells. In the essential gene screen, we identified a set of 1280 Anopheles genes that are highly enriched for genes involved in fundamental cell processes. For the clodronate liposome screen, we identified several candidate resistance factors and confirm their roles in the uptake and processing of clodronate liposomes through in vivo validation in Anopheles gambiae, providing new mechanistic detail of phagolysosome formation and clodronate liposome function. In summary, we demonstrate the application of a genome-wide CRISPR knockout platform in a major malaria vector and the identification of genes that are important for fitness and immune-related processes.

genetics↗

bioRxiv2024

Higher resolution pooled genome-wide CRISPR knockout screening in Drosophila cells using integration and anti-CRISPR (IntAC)

CRISPR screens enable systematic, scalable genotype-to-phenotype mapping. We previously developed a pooled CRISPR screening method for Drosophila melanogaster and mosquito cell lines using plasmid transfection and site-specific integration to introduce single guide (sgRNA) libraries, followed by PCR and sequencing of integrated sgRNAs. While effective, the method relies on early constitutive Cas9 activity that potentially can lead to discrepancies between genome edits and sgRNAs detected by PCR, reducing screen accuracy. To address this issue, we introduce a new method to co-transfect a plasmid expressing the anti-CRISPR protein AcrIIa4 to suppress Cas9 activity during early sgRNA expression, which we term "IntAC" (integrase with anti-CRISPR). IntAC allowed us to construct a new CRISPR screening approach driven by the high strength dU6:3 promoter. This new library dramatically improved precision-recall of fitness genes across the genome, retrieving 90-95% of essential gene groups within 5% error, allowing us to generate the most comprehensive list of cell fitness genes yet assembled for Drosophila. Our analysis determined that elevated sgRNA levels, made permissible by the IntAC approach, drove much of the improvement. The Drosophila fitness genes show strong correlation with human fitness genes and underscore the effects of paralogs on gene essentiality. We further demonstrate that IntAC combined with a targeted sgRNA sub-library enabled precise positive selection of a transporter under solute overload. IntAC represents a straightforward enhancement to existing Drosophila CRISPR screening methods, dramatically increasing accuracy, and might also be broadly applicable to virus-free CRISPR screens in other cell types, including mosquito, lepidopteran, tick, and mammalian cells.

genetics↗

bioRxiv2024

Enhanced Protein-Protein Interaction Discovery via AlphaFold-Multimer

Accurately mapping protein-protein interactions (PPIs) is critical for elucidating cellular functions and has significant implications for health and disease. Conventional experimental approaches, while foundational, often fall short in capturing direct, dynamic interactions, especially those with transient or small interfaces. Our study leverages AlphaFold-Multimer (AFM) to re-evaluate high-confidence PPI datasets from Drosophila and human. Our analysis uncovers a significant limitation of the AFM-derived interface pTM (ipTM) metric, which, while reflective of structural integrity, can miss physiologically relevant interactions at small interfaces or within flexible regions. To bridge this gap, we introduce the Local Interaction Score (LIS), derived from AFMs Predicted Aligned Error (PAE), focusing on areas with low PAE values, indicative of the high confidence in interaction predictions. The LIS method demonstrates enhanced sensitivity in detecting PPIs, particularly among those that involve flexible and small interfaces. By applying LIS to large-scale Drosophila datasets, we enhance the detection of direct interactions. Moreover, we present FlyPredictome, an online platform that integrates our AFM-based predictions with additional information such as gene expression correlations and subcellular localization predictions. This study not only improves upon AFMs utility in PPI prediction but also highlights the potential of computational methods to complement and enhance experimental approaches in the identification of PPI networks.

bioinformatics↗

bioRxiv2023

Genetic manipulation of an Ixodes scapularis cell line

Although genetic manipulation is one of the hallmarks in model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the blacklegged tick Ixodes scapularis, an arthropod that serves as a vector of several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology dependent recombination. Targeting the E3 ubiquitin ligase X-linked inhibitor of apoptosis (xiap) and its substrate p47 led to alteration in molecular signaling within the immune deficiency (IMD) network and increased infection of the rickettsial agent Anaplasma phagocytophilum in I. scapularis ISE6 cells. Collectively, our findings complement techniques for genetic engineering of ticks in vivo and aid in circumventing the long-life cycle of I. scapularis, of which limits efficient and scalable molecular genetic screens. ImportanceGenetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology dependent recombination. We ablated the expression of xiap and p47, two signaling molecules present in the immune deficiency (IMD) pathway of I. scapularis. Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agent A. phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the blacklegged tick Ixodes scapularis.

genetics↗

bioRxiv2023

Continuous muscle, glial, epithelial, neuronal, and hemocyte cell lines for Drosophila research

Expression of activated Ras, RasV12, provides Drosophila cultured cells with a proliferation and survival advantage that simplifies the generation of continuous cell lines. Here we used lineage restricted RasV12 expression to generate continuous cell lines of muscle, glial, and epithelial cell type. Additionally, cell lines with neuronal and hemocyte characteristics were isolated by cloning from cell cultures established with broad RasV12 expression. Differentiation with the hormone ecdysone caused maturation of cells from mesoderm lines into active muscle tissue and enhanced dendritic features in neuronal-like lines. Transcriptome analysis showed expression of key cell-type specific genes and the expected alignment with single cell sequencing data in several cases. Overall, the technique has produced in vitro cell models with characteristics of glia, epithelium, muscle, nerve, and hemocyte. The cells and associated data are available from the Drosophila Genomic Resource Center.

cell biology↗

bioRxiv2021

A genome-wide CRISPR screen identifies the glycosylation enzyme DPM1 as a modifier of DPAGT1 deficiency and ER stress

Partial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1-CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually cause CDGs. While both in vivo models ostensibly cause ER stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress.

genetics↗

bioRxiv2021

Identification of two pathways mediating protein targeting from ER to lipid droplets

Pathways localizing proteins to their sites of action within a cell are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, little is known about how proteins, such as key metabolic enzymes, target from the ER to cellular lipid droplets (LDs). Here, we identify two distinct pathways for ER-to-LD (ERTOLD) protein targeting: early ERTOLD, occurring during LD formation, and late ERTOLD, targeting mature LDs after their formation. By using systematic, unbiased approaches, we identified specific membrane-fusion machinery, including regulators, a tether, and SNARE proteins, that are required for late ERTOLD targeting. Components of this fusion machinery localize to LD-ER interfaces and appear to be organized at ER exit sites (ERES) to generate ER-LD membrane bridges. We also identified multiple cargoes for early and late ERTOLD. Collectively, our data provide a new model for how proteins target LDs from the ER. HIGHLIGHTSO_LIProteins localize to LDs either during formation or later through ER-LD bridges C_LIO_LISpecific membrane fusion machinery localizes to LDs and mediates protein targeting C_LIO_LISpecific ER exit site proteins associate with LDs and participate in ERTOLD targeting C_LIO_LIProteomic studies reveal cargoes for early and late ERTOLD targeting C_LI

cell biology↗

bioRxiv2021

Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

Mosquito-borne diseases present a worldwide public health burden. Genome-scale screening tools that could inform our understanding of mosquitos and their control are lacking. Here, we adapt a recombination-mediated cassette exchange system for delivery of CRISPR sgRNA libraries into cell lines from several mosquito species and perform pooled CRISPR screens in an Anopheles cell line. To implement this method, we engineered modified mosquito cell lines, validated promoters and developed bioinformatics tools for multiple mosquito species.

genetics↗

bioRxiv2020

BioLitMine: advanced mining of biomedical and biological literature about human genes and genes from major model organisms

The accumulation of biological and biomedical literature outpaces the ability of most researchers and clinicians to stay abreast of their own immediate fields, let alone a broader range of topics. Although available search tools support identification of relevant literature, finding relevant and key publications is not always straightforward. For example, important publications might be missed in searches with an official gene name due to gene synonyms. Moreover, ambiguity of gene names can result in retrieval of a large number of irrelevant publications. To address these issues and help researchers and physicians quickly identify relevant publications, we developed BioLitMine, an advanced literature mining tool that takes advantage of the medical subject heading (MeSH) index and gene-to-publication annotations already available for PubMed literature. Using BioLitMine, a user can identify what MeSH terms are represented in the set of publications associated with a given gene of the interest, or start with a term and identify relevant publications. Users can also use the tool to find co-cited genes and a build a literature co-citation network. In addition, BioLitMine can help users build a gene list relevant to a MeSH terms, such as a list of genes relevant to "stem cells" or "breast neoplasms." Users can also start with a gene or pathway of interest and identify authors associated with that gene or pathway, a feature that makes it easier to identify experts who might serve as collaborators or reviewers. Altogether, BioLitMine extends the value of PubMed-indexed literature and its existing expert curation by providing a robust and gene-centric approach to retrieval of relevant information.

bioinformatics↗

bioRxiv2019

Large-scale transgenic Drosophila resource collections for loss- and gain-of-function studies

The Transgenic RNAi Project (TRiP), a Drosophila functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNAi fly stocks. To date, the TRiP has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express sgRNAs targeting upstream of a gene transcription start site. Gene activation is triggered by co-expression of catalytically dead Cas9 (dCas9) fused to an activator domain, either VP64-p65-Rta (VPR) or Synergistic Activation Mediator (SAM). TRiP-KO stocks express one or two sgRNAs targeting the coding sequence of a gene or genes, allowing for generation of indels in both germline and somatic tissue. To date, we have generated more than 5,000 CRISPR-OE or -KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.

genetics↗

bioRxiv2019

SNP-CRISPR: a web tool for SNP-specific genome editing

CRISPR-Cas9 is a powerful genome editing technology in which a single guide RNA (sgRNA) confers target site specificity to achieve Cas9-mediated genome editing. Numerous sgRNA design tools have been developed based on reference genomes for humans and model organisms. However, existing resources are not optimal as genetic mutations or single nucleotide polymorphisms (SNPs) within the targeting region affect the efficiency of CRISPR-based approaches by interfering with guide-target complementarity. To facilitate identification of sgRNAs (1) in non-reference genomes, (2) across varying genetic backgrounds, or (3) for specific targeting of SNP-containing alleles, for example, disease relevant mutations, we developed a web tool, SNP-CRISPR (https://www.flyrnai.org/tools/snp_crispr/). SNP-CRISPR can be used to design sgRNAs based on public variant data sets or user-identified variants. In addition, the tool computes efficiency and specificity scores for sgRNA designs targeting both the variant and the reference. Moreover, SNP-CRISPR provides the option to upload multiple SNPs and target single or multiple nearby base changes simultaneously with a single sgRNA design. Given these capabilities, SNP-CRISPR has a wide range of potential research applications in model systems and for design of sgRNAs for disease-associated variant correction.

bioinformatics↗

bioRxiv2019

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms

We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018b). The method relied on double stranded DNA (dsDNA) homology donors with [~]1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.

genomics↗