Publications by authors named "Ethan Bier"

66 Publications

CopyCatchers are versatile active genetic elements that detect and quantify inter-homolog somatic gene conversion.

Nat Commun 2021 05 11;12(1):2625. Epub 2021 May 11.

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA.

CRISPR-based active genetic elements, or gene-drives, copied via homology-directed repair (HDR) in the germline, are transmitted to progeny at super-Mendelian frequencies. Active genetic elements also can generate widespread somatic mutations, but the genetic basis for such phenotypes remains uncertain. It is generally assumed that such somatic mutations are generated by non-homologous end-joining (NHEJ), the predominant double stranded break repair pathway active in somatic cells. Here, we develop CopyCatcher systems in Drosophila to detect and quantify somatic gene conversion (SGC) events. CopyCatchers inserted into two independent genetic loci reveal unexpectedly high rates of SGC in the Drosophila eye and thoracic epidermis. Focused RNAi-based genetic screens identify several unanticipated loci altering SGC efficiency, one of which (c-MYC), when downregulated, promotes SGC mediated by both plasmid and homologous chromosome-templates in human HEK293T cells. Collectively, these studies suggest that CopyCatchers can serve as effective discovery platforms to inform potential gene therapy strategies.
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http://dx.doi.org/10.1038/s41467-021-22927-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113449PMC
May 2021

Driving to Safety: CRISPR-Based Genetic Approaches to Reducing Antibiotic Resistance.

Trends Genet 2021 Mar 18. Epub 2021 Mar 18.

Tata Institute for Genetics and Society, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0349, USA; Collaborative to Halt Antibiotic-Resistant Microbes, Department of Pediatrics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0687, USA; Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0687, USA.

Bacterial resistance to antibiotics has reached critical levels, skyrocketing in hospitals and the environment and posing a major threat to global public health. The complex and challenging problem of reducing antibiotic resistance (AR) requires a network of both societal and science-based solutions to preserve the most lifesaving pharmaceutical intervention known to medicine. In addition to developing new classes of antibiotics, it is essential to safeguard the clinical efficacy of existing drugs. In this review, we examine the potential application of novel CRISPR-based genetic approaches to reducing AR in both environmental and clinical settings and prolonging the utility of vital antibiotics.
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http://dx.doi.org/10.1016/j.tig.2021.02.007DOI Listing
March 2021

Inherently confinable split-drive systems in Drosophila.

Nat Commun 2021 03 5;12(1):1480. Epub 2021 Mar 5.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA.

CRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences that restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.
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http://dx.doi.org/10.1038/s41467-021-21771-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935863PMC
March 2021

Hidden genomic features of an invasive malaria vector, Anopheles stephensi, revealed by a chromosome-level genome assembly.

BMC Biol 2021 02 10;19(1):28. Epub 2021 Feb 10.

Department of Ecology and Evolutionary Biology, University of California, Irvine, CA, 92697, USA.

Background: The mosquito Anopheles stephensi is a vector of urban malaria in Asia that recently invaded Africa. Studying the genetic basis of vectorial capacity and engineering genetic interventions are both impeded by limitations of a vector's genome assembly. The existing assemblies of An. stephensi are draft-quality and contain thousands of sequence gaps, potentially missing genetic elements important for its biology and evolution.

Results: To access previously intractable genomic regions, we generated a reference-grade genome assembly and full transcript annotations that achieve a new standard for reference genomes of disease vectors. Here, we report novel species-specific transposable element (TE) families and insertions in functional genetic elements, demonstrating the widespread role of TEs in genome evolution and phenotypic variation. We discovered 29 previously hidden members of insecticide resistance genes, uncovering new candidate genetic elements for the widespread insecticide resistance observed in An. stephensi. We identified 2.4 Mb of the Y chromosome and seven new male-linked gene candidates, representing the most extensive coverage of the Y chromosome in any mosquito. By tracking full-length mRNA for > 15 days following blood feeding, we discover distinct roles of previously uncharacterized genes in blood metabolism and female reproduction. The Y-linked heterochromatin landscape reveals extensive accumulation of long-terminal repeat retrotransposons throughout the evolution and degeneration of this chromosome. Finally, we identify a novel Y-linked putative transcription factor that is expressed constitutively throughout male development and adulthood, suggesting an important role.

Conclusion: Collectively, these results and resources underscore the significance of previously hidden genomic elements in the biology of malaria mosquitoes and will accelerate the development of genetic control strategies of malaria transmission.
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http://dx.doi.org/10.1186/s12915-021-00963-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876825PMC
February 2021

Application of the Relationship-Based Model to Engagement for Field Trials of Genetically Engineered Malaria Vectors.

Am J Trop Med Hyg 2020 Dec 21. Epub 2020 Dec 21.

Department of Molecular Biology and Biochemistry, University of California, Irvine, California.

The transition of new technologies for public health from laboratory to field is accompanied by a broadening scope of engagement challenges. Recent developments of vector control strategies involving genetically engineered mosquitoes with gene drives to assist in the eradication of malaria have drawn significant attention. Notably, questions have arisen surrounding community and regulatory engagement activities and of the need for examples of models or frameworks that can be applied to guide engagement. A relationship-based model (RBM) provides a framework that places stakeholders and community members at the center of decision-making processes, rather than as recipients of predetermined strategies, methods, and definitions. Successful RBM application in the transformation of healthcare delivery has demonstrated the importance of open dialogue and relationship development in establishing an environment where individuals are actively engaged in decision-making processes regarding their health. Although guidelines and recommendations for engagement for gene drives have recently been described, we argue here that communities and stakeholders should lead the planning, development, and implementation phases of engagement. The RBM provides a new approach to the development of ethical, transparent, and effective engagement strategies for malaria control programs.
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http://dx.doi.org/10.4269/ajtmh.20-0868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941841PMC
December 2020

Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi.

Nat Commun 2020 11 3;11(1):5553. Epub 2020 Nov 3.

Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, 92697-3900, USA.

Cas9/gRNA-mediated gene-drive systems have advanced development of genetic technologies for controlling vector-borne pathogen transmission. These technologies include population suppression approaches, genetic analogs of insecticidal techniques that reduce the number of insect vectors, and population modification (replacement/alteration) approaches, which interfere with competence to transmit pathogens. Here, we develop a recoded gene-drive rescue system for population modification of the malaria vector, Anopheles stephensi, that relieves the load in females caused by integration of the drive into the kynurenine hydroxylase gene by rescuing its function. Non-functional resistant alleles are eliminated via a dominantly-acting maternal effect combined with slower-acting standard negative selection, and rare functional resistant alleles do not prevent drive invasion. Small cage trials show that single releases of gene-drive males robustly result in efficient population modification with ≥95% of mosquitoes carrying the drive within 5-11 generations over a range of initial release ratios.
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http://dx.doi.org/10.1038/s41467-020-19426-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609566PMC
November 2020

Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives.

Mol Cell 2020 10 18;80(2):246-262.e4. Epub 2020 Sep 18.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA; Tata Institute for Genetics and Society, University of California, San Diego, La Jolla, CA, USA. Electronic address:

CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.
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http://dx.doi.org/10.1016/j.molcel.2020.09.003DOI Listing
October 2020

Translating gene drive science to promote linguistic diversity in community and stakeholder engagement.

Glob Public Health 2020 10 26;15(10):1551-1565. Epub 2020 Jun 26.

The Qualcomm Institute, Calit2, UC San Diego, La Jolla, CA, USA.

Information about genetic engineering (GE) for vector control in the United States is disseminated primarily in English, though non-English speakers are equally, and in some geographic regions even more affected by such technologies. Non-English-speaking publics should have equal access to such information, which is especially critical when the technology in question may impact whole communities. We convened an interdisciplinary workgroup to translate previously developed narrated slideshows on gene drive mosquitoes from English into Spanish, reviewing each iteration for scientific accuracy and accessibility to laypeople. Using the finalised stimuli, we conducted five online, chat-based focus groups with Spanish-speaking adults from California. Overall, participants expressed interest in the topic and were able to summarise the information presented in their own words. Importantly, participants asked for clarification and expressed scepticism about the information presented, indicating critical engagement with the material. Through collaboration with Spanish-speaking scientists engaged in the development of GE methods of vector control, we translated highly technical scientific information into Spanish that successfully engaged Spanish-speaking participants in conversations about this topic. In this manuscript, we document the feasibility of consulting Spanish-speaking publics about a complex emerging technology by drawing on the linguistic diversity of the scientific teams developing the technology.
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http://dx.doi.org/10.1080/17441692.2020.1779328DOI Listing
October 2020

A Drosophila Model for Clostridium difficile Toxin CDT Reveals Interactions with Multiple Effector Pathways.

iScience 2020 Feb 25;23(2):100865. Epub 2020 Jan 25.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093-0335, USA; Tata Institute for Genetics and Society-UCSD, La Jolla, CA 92093-0335, USA. Electronic address:

Clostridium difficile infections (CDIs) cause severe and occasionally life-threatening diarrhea. Hyper-virulent strains produce CDT, a toxin that ADP-ribosylates actin monomers and inhibits actin polymerization. We created transgenic Drosophila lines expressing the catalytic subunit CDTa to investigate its interaction with host signaling pathways in vivo. When expressed in the midgut, CDTa reduces body weight and fecal output and compromises survival, suggesting severe impairment of digestive functions. At the cellular level, CDTa induces F-actin network collapse, elimination of the intestinal brush border, and disruption of intercellular junctions. We confirm toxin-dependent re-distribution of Rab11 to enterocytes' apical surface and observe suppression of CDTa phenotypes by a Dominant-Negative form of Rab11 or RNAi of the dedicated Rab11GEF Crag (DENND4). We also report that Calmodulin (Cam) is required to mediate CDTa activity. In parallel, chemical inhibition of the Cam/Calcineurin pathway by Cyclosporin A or FK506 also reduces CDTa phenotypes, potentially opening new avenues for treating CDIs.
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http://dx.doi.org/10.1016/j.isci.2020.100865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7011083PMC
February 2020

A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment.

Nat Commun 2020 01 17;11(1):352. Epub 2020 Jan 17.

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, 92093, USA.

CRISPR-based gene drives can spread through wild populations by biasing their own transmission above the 50% value predicted by Mendelian inheritance. These technologies offer population-engineering solutions for combating vector-borne diseases, managing crop pests, and supporting ecosystem conservation efforts. Current technologies raise safety concerns for unintended gene propagation. Herein, we address such concerns by splitting the drive components, Cas9 and gRNAs, into separate alleles to form a trans-complementing split-gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This dual-component configuration allows for combinatorial transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a small-molecule-controlled version to investigate the biology of component inheritance and resistant allele formation, and to study the effects of maternal inheritance and impaired homology on efficiency. Lastly, mathematical modeling of tGD spread within populations reveals potential advantages for improving current gene-drive technologies for field population modification.
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http://dx.doi.org/10.1038/s41467-019-13977-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969112PMC
January 2020

Author Correction: Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline.

Nature 2020 01;577(7792):E8

Division of Biological Sciences, Section of Cellular and Developmental Biology, University of California, San Diego, La Jolla, CA, USA.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41586-019-1861-4DOI Listing
January 2020

Assessment of a Split Homing Based Gene Drive for Efficient Knockout of Multiple Genes.

G3 (Bethesda) 2020 02 6;10(2):827-837. Epub 2020 Feb 6.

Section of Cell and Developmental Biology and

Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent disease transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate genes has not been thoroughly explored. To test this approach, we generated a split-HGD in that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host-encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of both genes during super-Mendelian propagation of split-HGD. Furthermore, both genes were knocked out one generation earlier than expected indicating the robust somatic expression of Cas9 driven by germline-limited promoters. We also assess the efficiency of 'shadow drive' generated by maternally deposited Cas9 protein and accumulation of drive-induced resistance alleles along multiple generations, and discuss design principles of HGD that could mitigate the accumulation of resistance alleles while incorporating a GME.
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http://dx.doi.org/10.1534/g3.119.400985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003086PMC
February 2020

A bacterial gene-drive system efficiently edits and inactivates a high copy number antibiotic resistance locus.

Nat Commun 2019 12 16;10(1):5726. Epub 2019 Dec 16.

Tata Institute for Genetics and Society, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0349, USA.

Gene-drive systems in diploid organisms bias the inheritance of one allele over another. CRISPR-based gene-drive expresses a guide RNA (gRNA) into the genome at the site where the gRNA directs Cas9-mediated cleavage. In the presence of Cas9, the gRNA cassette and any linked cargo sequences are copied via homology-directed repair (HDR) onto the homologous chromosome. Here, we develop an analogous CRISPR-based gene-drive system for the bacterium Escherichia coli that efficiently copies a gRNA cassette and adjacent cargo flanked with sequences homologous to the targeted gRNA/Cas9 cleavage site. This "pro-active" genetic system (Pro-AG) functionally inactivates an antibiotic resistance marker on a high copy number plasmid with ~ 100-fold greater efficiency than control CRISPR-based methods, suggesting an amplifying positive feedback loop due to increasing gRNA dosage. Pro-AG can likewise effectively edit large plasmids or single-copy genomic targets or introduce functional genes, foreshadowing potential applications to biotechnology or biomedicine.
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http://dx.doi.org/10.1038/s41467-019-13649-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6915771PMC
December 2019

Efficient allelic-drive in Drosophila.

Nat Commun 2019 04 9;10(1):1640. Epub 2019 Apr 9.

Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0335, USA.

Gene-drive systems developed in several organisms result in super-Mendelian inheritance of transgenic insertions. Here, we generalize this "active genetic" approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. We test two configurations for allelic-drive: one, copy-cutting, in which a non-preferred allele is selectively targeted for Cas9/guide RNA (gRNA) cleavage, and a more general approach, copy-grafting, that permits selective inheritance of a desired allele located in close proximity to the gRNA cut site. We also characterize a phenomenon we refer to as lethal-mosaicism that dominantly eliminates NHEJ-induced mutations and favors inheritance of functional cleavage-resistant alleles. These two efficient allelic-drive methods, enhanced by lethal mosaicism and a trans-generational drive process we refer to as "shadow-drive", have broad practical applications in improving health and agriculture and greatly extend the active genetics toolbox.
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http://dx.doi.org/10.1038/s41467-019-09694-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456580PMC
April 2019

Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline.

Nature 2019 02 23;566(7742):105-109. Epub 2019 Jan 23.

Division of Biological Sciences, Section of Cellular and Developmental Biology, University of California, San Diego, La Jolla, CA, USA.

A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR-Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR-Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR-Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.
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http://dx.doi.org/10.1038/s41586-019-0875-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6367021PMC
February 2019

N-linked glycosylation restricts the function of Short gastrulation to bind and shuttle BMPs.

Development 2018 11 19;145(22). Epub 2018 Nov 19.

Institute for Biomedical Sciences, Federal University of Rio de Janeiro, RJ, Brazil, 21941-902

Disorders of N-linked glycosylation are increasingly reported in the literature. However, the targets that are responsible for the associated developmental and physiological defects are largely unknown. Bone morphogenetic proteins (BMPs) act as highly dynamic complexes to regulate several functions during development. The range and strength of BMP activity depend on interactions with glycosylated protein complexes in the extracellular milieu. Here, we investigate the role of glycosylation for the function of the conserved extracellular BMP antagonist Short gastrulation (Sog). We identify conserved N-glycosylated sites and describe the effect of mutating these residues on BMP pathway activity in Functional analysis reveals that loss of individual Sog glycosylation sites enhances BMP antagonism and/or increases the spatial range of Sog effects in the tissue. Mechanistically, we provide evidence that N-terminal and stem glycosylation controls extracellular Sog levels and distribution. The identification of similar residues in vertebrate Chordin proteins suggests that N-glycosylation may be an evolutionarily conserved process that adds complexity to the regulation of BMP activity.
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http://dx.doi.org/10.1242/dev.167338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6262792PMC
November 2018

Innate Immune Interactions between and Host Neutrophils.

Front Cell Infect Microbiol 2018 22;8. Epub 2018 Jan 22.

Division of Host-Microbe Systems and Therapeutics, Department of Pediatrics, University of California, San Diego, San Diego, CA, United States.

, the causative agent of anthrax, has been a focus of study in host-pathogen dynamics since the nineteenth century. While the interaction between anthrax and host macrophages has been extensively modeled, comparatively little is known about the effect of anthrax on the immune function of neutrophils, a key frontline effector of innate immune defense. Here we showed that depletion of neutrophils significantly enhanced mortality in a systemic model of anthrax infection in mice. , we found that freshly isolated human neutrophils can rapidly kill anthrax, with specific inhibitor studies showing that phagocytosis and reactive oxygen species (ROS) generation contribute to this efficient bacterial clearance. Anthrax toxins, comprising lethal toxin (LT) and edema toxin (ET), are known to have major roles in macrophage resistance and systemic toxicity. Employing isogenic wild-type and mutant toxin-deficient strains, we show that despite previous studies that reported inhibition of neutrophil function by purified LT or ET, endogenous production of these toxins by live vegetative failed to alter key neutrophil functions. The lack of alteration in neutrophil function is accompanied by rapid killing of by neutrophils, regardless of the bacteria's expression of anthrax toxins. Lastly, our study demonstrates for the first time that anthrax induced neutrophil extracellular trap (NET) formation.
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http://dx.doi.org/10.3389/fcimb.2018.00002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5786542PMC
January 2019

Advances in Engineering the Fly Genome with the CRISPR-Cas System.

Genetics 2018 01;208(1):1-18

Biochemistry Department, University of Wisconsin-Madison, Wisconsin 53706

has long been a premier model for the development and application of cutting-edge genetic approaches. The CRISPR-Cas system now adds the ability to manipulate the genome with ease and precision, providing a rich toolbox to interrogate relationships between genotype and phenotype, to delineate and visualize how the genome is organized, to illuminate and manipulate RNA, and to pioneer new gene drive technologies. Myriad transformative approaches have already originated from the CRISPR-Cas system, which will likely continue to spark the creation of tools with diverse applications. Here, we provide an overview of how CRISPR-Cas gene editing has revolutionized genetic analysis in and highlight key areas for future advances.
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http://dx.doi.org/10.1534/genetics.117.1113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753851PMC
January 2018

CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous -L2 CRM reveals unexpected complexity.

Elife 2017 12 23;6. Epub 2017 Dec 23.

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California.

The () locus encodes transcription factors required for induction of the L2 wing vein in . Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for expression in the L2 vein primordium. Phenotypic analysis of these '' mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis.
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http://dx.doi.org/10.7554/eLife.30281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800851PMC
December 2017

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport.

PLoS Pathog 2017 Sep 25;13(9):e1006603. Epub 2017 Sep 25.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, United States of America.

Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies.
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http://dx.doi.org/10.1371/journal.ppat.1006603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612732PMC
September 2017

Influenza NS1 directly modulates Hedgehog signaling during infection.

PLoS Pathog 2017 Aug 24;13(8):e1006588. Epub 2017 Aug 24.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, California, United States of America.

The multifunctional NS1 protein of influenza A viruses suppresses host cellular defense mechanisms and subverts other cellular functions. We report here on a new role for NS1 in modifying cell-cell signaling via the Hedgehog (Hh) pathway. Genetic epistasis experiments and FRET-FLIM assays in Drosophila suggest that NS1 interacts directly with the transcriptional mediator, Ci/Gli1. We further confirmed that Hh target genes are activated cell-autonomously in transfected human lung epithelial cells expressing NS1, and in infected mouse lungs. We identified a point mutation in NS1, A122V, that modulates this activity in a context-dependent fashion. When the A122V mutation was incorporated into a mouse-adapted influenza A virus, it cell-autonomously enhanced expression of some Hh targets in the mouse lung, including IL6, and hastened lethality. These results indicate that, in addition to its multiple intracellular functions, NS1 also modifies a highly conserved signaling pathway, at least in part via cell autonomous activities. We discuss how this new Hh modulating function of NS1 may influence host lethality, possibly through controlling cytokine production, and how these new insights provide potential strategies for combating infection.
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http://dx.doi.org/10.1371/journal.ppat.1006588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587344PMC
August 2017

The dawn of active genetics.

Bioessays 2016 Jan 10;38(1):50-63. Epub 2015 Dec 10.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA, USA.

On December 18, 2014, a yellow female fly quietly emerged from her pupal case. What made her unique was that she had only one parent carrying a mutant allele of this classic recessive locus. Then, one generation later, after mating with a wild-type male, all her offspring displayed the same recessive yellow phenotype. Further analysis of other such yellow females revealed that the construct causing the mutation was converting the opposing chromosome with 95% efficiency. These simple results, seen also in mosquitoes and yeast, open the door to a new era of genetics wherein the laws of traditional Mendelian inheritance can be bypassed for a broad variety of purposes. Here, we consider the implications of this fundamentally new form of "active genetics," its applications for gene drives, reversal and amplification strategies, its potential for contributing to cell and gene therapy strategies, and ethical/biosafety considerations associated with such active genetic elements. Also watch the Video Abstract.
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http://dx.doi.org/10.1002/bies.201500102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819344PMC
January 2016

Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi.

Proc Natl Acad Sci U S A 2015 Dec 23;112(49):E6736-43. Epub 2015 Nov 23.

Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900; Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697-4500

Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼ 17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼ 99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.
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http://dx.doi.org/10.1073/pnas.1521077112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679060PMC
December 2015

EMBRYO DEVELOPMENT. BMP gradients: A paradigm for morphogen-mediated developmental patterning.

Science 2015 Jun;348(6242):aaa5838

Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, CA 90095-1662, USA. Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA 90095-1662, USA.

Bone morphogenetic proteins (BMPs) act in dose-dependent fashion to regulate cell fate choices in a myriad of developmental contexts. In early vertebrate and invertebrate embryos, BMPs and their antagonists establish epidermal versus central nervous system domains. In this highly conserved system, BMP antagonists mediate the neural-inductive activities proposed by Hans Spemann and Hilde Mangold nearly a century ago. BMPs distributed in gradients subsequently function as morphogens to subdivide the three germ layers into distinct territories and act to organize body axes, regulate growth, maintain stem cell niches, or signal inductively across germ layers. In this Review, we summarize the variety of mechanisms that contribute to generating reliable developmental responses to BMP gradients and other morphogen systems.
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http://dx.doi.org/10.1126/science.aaa5838DOI Listing
June 2015

Genome editing. The mutagenic chain reaction: a method for converting heterozygous to homozygous mutations.

Science 2015 Apr 19;348(6233):442-4. Epub 2015 Mar 19.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92095, USA.

An organism with a single recessive loss-of-function allele will typically have a wild-type phenotype, whereas individuals homozygous for two copies of the allele will display a mutant phenotype. We have developed a method called the mutagenic chain reaction (MCR), which is based on the CRISPR/Cas9 genome-editing system for generating autocatalytic mutations, to produce homozygous loss-of-function mutations. In Drosophila, we found that MCR mutations efficiently spread from their chromosome of origin to the homologous chromosome, thereby converting heterozygous mutations to homozygosity in the vast majority of somatic and germline cells. MCR technology should have broad applications in diverse organisms.
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http://dx.doi.org/10.1126/science.aaa5945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687737PMC
April 2015

Hedgehog: linking uracil to innate defense.

Cell Host Microbe 2015 Feb;17(2):146-8

Department of Pediatrics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0687, USA; Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0687, USA. Electronic address:

The ability of the gut epithelium to defend against pathogens while tolerating harmless commensal organisms remains an important puzzle. In this issue of Cell Host & Microbe, Lee et al. (2015) reveal how pathogen-secreted uracil acts at two steps to induce ROS via the Hedgehog pathway.
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http://dx.doi.org/10.1016/j.chom.2015.01.010DOI Listing
February 2015

BMPs regulate msx gene expression in the dorsal neuroectoderm of Drosophila and vertebrates by distinct mechanisms.

PLoS Genet 2014 Sep 11;10(9):e1004625. Epub 2014 Sep 11.

Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, California, United States of America.

In a broad variety of bilaterian species the trunk central nervous system (CNS) derives from three primary rows of neuroblasts. The fates of these neural progenitor cells are determined in part by three conserved transcription factors: vnd/nkx2.2, ind/gsh and msh/msx in Drosophila melanogaster/vertebrates, which are expressed in corresponding non-overlapping patterns along the dorsal-ventral axis. While this conserved suite of "neural identity" gene expression strongly suggests a common ancestral origin for the patterning systems, it is unclear whether the original regulatory mechanisms establishing these patterns have been similarly conserved during evolution. In Drosophila, genetic evidence suggests that Bone Morphogenetic Proteins (BMPs) act in a dosage-dependent fashion to repress expression of neural identity genes. BMPs also play a dose-dependent role in patterning the dorsal and lateral regions of the vertebrate CNS, however, the mechanism by which they achieve such patterning has not yet been clearly established. In this report, we examine the mechanisms by which BMPs act on cis-regulatory modules (CRMs) that control localized expression of the Drosophila msh and zebrafish (Danio rerio) msxB in the dorsal central nervous system (CNS). Our analysis suggests that BMPs act differently in these organisms to regulate similar patterns of gene expression in the neuroectoderm: repressing msh expression in Drosophila, while activating msxB expression in the zebrafish. These findings suggest that the mechanisms by which the BMP gradient patterns the dorsal neuroectoderm have reversed since the divergence of these two ancient lineages.
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http://dx.doi.org/10.1371/journal.pgen.1004625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161316PMC
September 2014

RAB11-mediated trafficking in host-pathogen interactions.

Nat Rev Microbiol 2014 Sep;12(9):624-34

Section of Cell and Developmental Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.

Many bacterial and viral pathogens block or subvert host cellular processes to promote successful infection. One host protein that is targeted by invading pathogens is the small GTPase RAB11, which functions in vesicular trafficking. RAB11 functions in conjunction with a protein complex known as the exocyst to mediate terminal steps in cargo transport via the recycling endosome to cell-cell junctions, phagosomes and cellular protrusions. These processes contribute to host innate immunity by promoting epithelial and endothelial barrier integrity, sensing and immobilizing pathogens and repairing pathogen-induced cellular damage. In this Review, we discuss the various mechanisms that pathogens have evolved to disrupt or subvert RAB11-dependent pathways as part of their infection strategy.
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http://dx.doi.org/10.1038/nrmicro3325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4274738PMC
September 2014