Search bioRxiv⌕ Search

Biology subjects

Holden, K.

Publications and source records attributed to Holden, K..

13 recordsLinked to original sources

Expression of non-neuronal Tau in humans and mice

BackgroundTau, encoded by the microtubule-associated protein tau gene (MAPT), is traditionally associated with neuronal function in maintaining cytoskeletal stability and regulating intracellular transport. Its dysregulation is implicated in a range of neurodegenerative disorders. Beyond its well-established high levels of expression in neurons of the central and peripheral nervous systems, increasing evidence indicates that Tau is also expressed in non-neuronal cells/tissues such as muscle, kidney, testis, and pancreas, albeit at much lower levels than in neurons. However, the assessment of low levels of non-neuronal Tau requires that the specificities of the anti-Tau antibodies used are fully validated. MethodsThree previously validated anti-Tau antibodies (Tau-12, Tau-1, RD3) were used to test Tau expression levels in 33 mouse and 66 human tissues using Food and Drug Administration-quality tissue microarrays in immunocytochemistry and western blot analyses. ResultsThe presence of Tau was confirmed in salivary gland, kidney, skeletal muscle, heart, pancreas and oesophagus in both human and mouse tissues. ConclusionsDysregulation of Tau protein expression and alterations in its post-translational modification is a causative factor in neurodegenerative disease. We have demonstrated a wider Tau expression in other tissues and cells, outside the brain, which may be dysregulated in other diseases.

cell biology↗

Enhanced RNA-targeting CRISPR-Cas technology in zebrafish

CRISPR-Cas13 systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity in mammalian cells and mouse models. Moreover, its efficiency could be improved in vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations to enhance the CRISPR-RfxCas13d system in zebrafish. We i) used chemically modified gRNAs to allow more penetrant loss-of-function phenotypes, ii) improved nuclear RNA-targeting, and iii) compared different computational models and determined the most accurate to predict gRNA activity in vivo. Furthermore, we demonstrated that transient CRISPR-RfxCas13d can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except when targeting extremely abundant and ectopic RNAs. Finally, we implemented alternative RNA-targeting CRISPR-Cas systems with reduced or absent collateral activity. Altogether, these findings contribute to CRISPR-Cas technology optimization for RNA targeting in zebrafish through transient approaches and assist in the progression of in vivo applications.

developmental biology↗

Rapid generation of precision preclinical cancer models using regulatable in vivo base editing

Single nucleotide variants (SNVs) comprise the majority of cancer-associated genetic changes and can have diverse effects on protein function. Despite a comprehensive catalogue of SNVs across human cancers, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a murine system for temporal and regulatable in vivo cytosine base editing (iBE). The iBE mice show robust, doxycycline-dependent expression across a broad range of tissues with no evidence of DNA or RNA off-target effects. Transient iBE induction drives efficient creation of individual or multiple SNVs in intestinal, lung, and pancreatic organoids, while temporal iBE regulation allows controlled sequential genome editing. Moreover, in situ delivery of plasmid-based or synthetic sgRNAs to target tissues facilitates the simple and rapid generation of pre-clinical cancer models. Overall, iBE is a powerful in vivo platform to define and interrogate the genetic drivers of cancer.

cancer biology↗

Controlling homology-directed repair outcomes in human stem cells with dCas9

Modeling human disease in human stem cells requires precise, scarless editing of single nucleotide variants (SNV) on one or both chromosomes. Here we describe improved conditions for Cas9 RNP editing of SNVs that yield high rates of biallelic homology-directed repair. To recover both heterozygous and homozygous SNV clones, catalytically inactive dCas9was added to moderate high activity Cas9 RNPs. dCas9 can also block re-cutting and damage to SNV alleles engineered with non-overlapping guide RNAs.

genetics↗

Viral E Protein Neutralizes BET Protein-Mediated Post-Entry Antagonism of SARS-CoV-2

Inhibitors of Bromodomain and Extra-terminal domain (BET) proteins are possible anti-SARS-CoV-2 prophylactics as they downregulate angiotensin-converting enzyme 2 (ACE2). Here, we show that BET proteins should not be inactivated therapeutically as they are critical antiviral factors at the post-entry level. Knockouts of BRD3 or BRD4 in cells overexpressing ACE2 exacerbate SARS-CoV-2 infection; the same is observed when cells with endogenous ACE2 expression are treated with BET inhibitors during infection, and not before. Viral replication and mortality are also enhanced in BET inhibitor-treated mice overexpressing ACE2. BET inactivation suppresses interferon production induced by SARS-CoV-2, a process phenocopied by the envelope (E) protein previously identified as a possible "histone mimetic." E protein, in an acetylated form, directly binds the second bromodomain of BRD4. Our data support a model where SARS-CoV-2 E protein evolved to antagonize interferon responses via BET protein inhibition; this neutralization should not be further enhanced with BET inhibitor treatment.

molecular biology↗

Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases

Programmable and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double-strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. We present Programmable Addition via Site-specific Targeting Elements, or PASTE, which achieves efficient and versatile gene integration at diverse loci by directing insertion with a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. Without generating double stranded breaks, we demonstrate integration of sequences as large as [~]36 kb with rates between 10-50% at multiple genomic loci across three human cell lines, primary T cells, and quiescent non-dividing primary human hepatocytes. To further improve PASTE, we discover thousands of novel serine integrases and cognate attachment sites from metagenomes and engineer active orthologs for high-efficiency integration using PASTE. We apply PASTE to fluorescent tagging of proteins, integration of therapeutically relevant genes, and production and secretion of transgenes. Leveraging the orthogonality of serine integrases, we engineer PASTE for multiplexed gene integration, simultaneously integrating three different genes at three genomic loci. PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in non-dividing cells and fewer detectable off-target events. For therapeutic applications, PASTE can be delivered as mRNA with synthetically modified guides to programmably direct insertion of DNA templates carried by AAV or adenoviral vectors. PASTE expands the capabilities of genome editing via drag-and-drop gene integration, offering a platform with wide applicability for research, cell engineering, and gene therapy. One Sentence SummaryA new technology combining CRISPR-mediated genome editing and site-specific integrases enables efficient programmable gene integration at any targeted genomic locus without double-strand DNA breaks, leading to broad applications in basic science research, cell engineering, and gene therapy.

bioengineering↗

Chemically modified guide RNAs enhance CRISPR-Cas13 knockdown in human cells

RNA-targeting CRISPR-Cas13 proteins have recently emerged as a powerful platform to transiently modulate gene expression outcomes. However, protein and CRISPR RNA (crRNA) delivery in human cells can be challenging and knockdown can be transient due to rapid crRNA degradation. Here we compare several chemical RNA modifications at different positions to identify synthetic crRNAs that improve RNA targeting efficiency and half-life in human cells. We show that co-delivery of modified crRNAs and recombinant Cas13 enzyme in ribonucleoprotein (RNP) complexes enables transient gene expression modulation in primary CD4+ and CD8+ T-cells. This system represents a robust and efficient method to transiently modulate transcripts without genetic manipulation.

bioengineering↗

Identification of DAXX As A Restriction Factor Of SARS-CoV-2 Through A CRISPR/Cas9 Screen

Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identified DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX was sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricted infection. In contrast with most of its previously described antiviral activities, DAXX-mediated restriction of SARS-CoV-2 was independent of the SUMOylation pathway. SARS-CoV-2 infection triggered the re-localization of DAXX to cytoplasmic sites and promoted its degradation. Mechanistically, this process was mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.

microbiology↗

BRD2 inhibition blocks SARS-CoV-2 infection in vitro by reducing transcription of the host cell receptor ACE2

SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. We found that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a novel therapeutic target for COVID-19.

cell biology↗

Phenotypic and environmental predictors of reproductive success in painted turtles

Sexual selection is often assumed to elicit sexually dimorphic traits. However, most work on this assumption in tetrapod vertebrates has focused on birds. In this field experiment, we assessed relationships between both sexually dimorphic (body size, claw length) and non-dimorphic traits (forelimb stripe color, baseline corticosterone concentrations) and reproductive success in adult painted turtles to explicate the roles of these phenotypes in mate choice and the evolution of sexual dimorphism. We also modified adult sex ratios in experimental ponds to elucidate the role of biased sex ratios on reproductive success, which is a timely test of the potential threat of biased sex ratios on population persistence in a species with temperature-dependent sex determination. We found no strong influence of male phenotypes on male siring success, but female body size and baseline corticosterone concentrations predicted female clutch sizes. We find weak evidence that adult sex ratio influences male siring success, with a male-biased sex ratio producing lower male siring success than a female-biased sex ratio. This study offers evidence that female mate choice may not be an important selective force on male phenotypes, but that instead selection occurs on female phenotypes, particularly body size and corticosterone concentrations. Further, biased adult sex ratios can influence reproductive success of both sexes. Finally, the use of Kompetitive Allele Specific PCR (KASP) was highly successful in parentage analysis, which adds reptiles to the growing list of taxa successfully genotyped with this new technology. Lay SummaryFemale painted turtles arent choosy about traits of their mates. In a field experiment, we find that male traits do not predict male fitness, but key female traits (body size and stress levels) do predict female reproductive success. Further, we find weak evidence that adult sex ratio influences individual fitness in this species with environmental sex determination. Ultimately, we reject the long-assumed importance of female mate choice in this freshwater turtle.

ecology↗

Prime Editing in Mice Reveals the Essentiality of a Single Base in Driving Tissue-Specific Gene Expression

Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Several genome editing platforms have emerged to evaluate the functionality of TFBS in animals. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice to demonstrate the essentiality of a single TFBS, called a CArG box, in the promoter region of the Tspan2 gene. HDR-mediated substitution of three base pairs in the Tspan2 CArG box resulted in 20/37 (54%) founder mice testing positive for the correct edit. Mice homozygous for this edit showed near loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Using the same protospacer, PE2-mediated editing of a single base in the Tspan2 CArG box yielded 12/47 (26%) founder mice testing positive for the correct edit. This single base substitution resulted in [~]90% loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Targeted sequencing demonstrated all PE2 and HDR founders with some frequency of on-target editing. However, whereas no spurious on-target indels were detected in any of the PE2 founders, many HDR founders showed variable levels of on-target indels. Further, off-target analysis by targeted sequencing revealed mutations in 5/11 (45%) HDR founders but none in PE2 founders. These results demonstrate high fidelity editing of a TFBS with PE2 and suggest a new paradigm for Cre/loxP-free tissue-specific gene inactivation via single base substitution in a TFBS. The PE2 platform of genome editing represents a powerful approach for modeling and correcting relevant noncoding SNVs in the mouse.

genetics↗

Functional genomic screens identify human host factors for SARS-CoV-2 and common cold coronaviruses

The Coronaviridae are a family of viruses that causes disease in humans ranging from mild respiratory infection to potentially lethal acute respiratory distress syndrome. Finding host factors that are common to multiple coronaviruses could facilitate the development of therapies to combat current and future coronavirus pandemics. Here, we conducted parallel genome-wide CRISPR screens in cells infected by SARS-CoV-2 as well as two seasonally circulating common cold coronaviruses, OC43 and 229E. This approach correctly identified the distinct viral entry factors ACE2 (for SARS-CoV-2), aminopeptidase N (for 229E) and glycosaminoglycans (for OC43). Additionally, we discovered phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis as critical host pathways supporting infection by all three coronaviruses. By contrast, the lysosomal protein TMEM106B appeared unique to SARS-CoV-2 infection. Pharmacological inhibition of phosphatidylinositol phosphate biosynthesis and cholesterol homeostasis reduced replication of all three coronaviruses. These findings offer important insights for the understanding of the coronavirus life cycle as well as the potential development of host-directed therapies.

microbiology↗

Inference of CRISPR Edits from Sanger Trace Data

Efficient precision genome editing requires a quick, quantitative, and inexpensive assay of editing outcomes. Here we present ICE (Inference of CRISPR Edits), which enables robust analysis of CRISPR edits using Sanger data. ICE proposes potential outcomes for editing with guide RNAs (gRNAs) and then determines which are supported by the data via regression. Additionally, we develop a score called ICE-D (Discordance) that can provide information on large or unexpected edits. We empirically confirm through over 1,800 edits that the ICE algorithm is robust, reproducible, and can analyze CRISPR experiments within days after transfection. We also confirm that ICE strongly correlates with next-generation sequencing of amplicons (Amp-Seq). The ICE tool is free to use and offers several improvements over current analysis tools. For instance, ICE can analyze individual experiments as well as multiple experiments simultaneously (batch analysis). ICE can also detect a wider variety of outcomes, including multi-guide edits (multiple gRNAs per target) and edits resulting from homology-directed repair (HDR), such as knock-ins and base edits. ICE is a reliable analysis tool that can significantly expedite CRISPR editing workflows. It is available online at ice.synthego.com, and the source code is at github.com/synthego-open/ice

bioinformatics↗