Testing non-autonomous antimalarial gene drive effectors using self-eliminating drivers in the African mosquito vector Anopheles gambiae

Gene drives for mosquito population modification are novel tools for malaria control. Strategies to safely test antimalarial effectors in the field are required. Here, we modified the Anopheles gambiae zpg locus to host a CRISPR/Cas9 integral gene drive allele (zpgD) and characterized its behaviour and resistance profile. We found that zpgD dominantly sterilizes females but can induce efficient drive at other loci when it itself encounters resistance. We combined zpgD with multiple previously characterized non-autonomous payload drives and found that, as zpgD self-eliminates, it leads to conversion of mosquito cage populations at these loci. Our results demonstrate how self-eliminating drivers could allow safe testing of non-autonomous effector-traits by local population modification. They also suggest that after engendering resistance, gene drives intended for population suppression could nevertheless serve to propagate subsequently released non-autonomous payload genes, allowing modification of vector populations initially targeted for suppression

A Systems-level study of giant regulation in Drosophila melanogaster

Esta tesis revela la regulación transcripcional del gen gap giant (gt) en el embrión blastodermal de Drosophila por ingeniería inversa: un modelo matemático infiere los mecanismos subyacentes de datos cuantitativos de expresión recopilados en un fondo genético silvestre. El modelo se amolda a mRNA reportero controlado por elementos reguladores en cis (CRE) de gt. Es una herramienta potente para investigar cómo se forma el patrón a nivel molecular por los sitios de unión de factores de transcripción y permite predecir la expresión en cepas mutantes. La presente tesis esclarece la regulación diferencial de dos CRE adyacentes de gt y presenta la primera evidencia experimental de auto-activación de gt mediante mutagénesis de sus elementos reguladores. Tras la optimización de los parámetros en un fondo de tipo silvestre, el modelo predice correctamente los cambios observados en mutantes de Krüppel y tailless. Otras contribuciones reglamentarias sugeridas por el modelo son confirmadas por la evaluación sistemática de los CREs en mutantesThis thesis unravels the transcriptional regulation of the gap gene giant (gt) in the Drosophila blastoderm embryo via a reverse-engineering approach: a mathematical model infers the underlying mechanisms from quantitative expression data collected in the wild-type background. The model is fit to reporter mRNA driven by cis-regulatory elements (CRE) of gt. It is a powerful tool to investigate how the pattern is formed at the molecular level from transcription factor binding sites and it gives us the ability to predict the expression in mutants. This thesis elucidates the differential regulation of two adjacent gt CREs and presents the first experimental evidence for Gt auto-activation via site-directed mutagenesis of its enhancers. After optimizing the parameters in the wild-type background, the model correctly predicts the observed changes in Krüppel and tailless mutants. Other regulatory contributions suggested by the model are confirmed by systematic evaluation of the CREs in mutant

Reverse-engineering post-transcriptional regulation of gap genes in Drosophila melanogaster

16 páginas, 6 figuras, 1 tablaSystems biology proceeds through repeated cycles of experiment and modeling. One way to implement this is reverse engineering, where models are fit to data to infer and analyse regulatory mechanisms. This requires rigorous methods to determine whether model parameters can be properly identified. Applying such methods in a complex biological context remains challenging. We use reverse engineering to study post-transcriptional regulation in pattern formation. As a case study, we analyse expression of the gap genes Krüppel, knirps, and giant in Drosophila melanogaster. We use detailed, quantitative datasets of gap gene mRNA and protein expression to solve and fit a model of post-transcriptional regulation, and establish its structural and practical identifiability. Our results demonstrate that post-transcriptional regulation is not required for patterning in this system, but is necessary for proper control of protein levels. Our work demonstrates that the uniqueness and specificity of a fitted model can be rigorously determined in the context of spatio-temporal pattern formation. This greatly increases the potential of reverse engineering for the study of development and other, similarly complex, biological processesThis collaborative project was carried out in the context of the BioPreDyn consortium, which is co-ordinated by JJ and JRB, and funded by European Commission grant FP7-KBBE-2011-5/289434. The laboratory of JJ is funded by the MEC-EMBL agreement for the EMBL/CRG Research Unit in Systems Biology. Additional financial support was provided by SGR Grant 406 from the Catalan funding agency AGAUR, and by grants BFU2009- 10184 and 273 BFU2009-09168 from the Spanish Ministerio de Economia y Competitividad (MINECO). The group at IIM-CSIC acknowledges financial support from MINECO and the European Regional Development Fund (ERDF; project “MultiScales”, DPI2011-28112-C04-03). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

A quantitative validated model reveals two phases of transcriptional regulation for the gap gene giant in Drosophila

Understanding eukaryotic transcriptional regulation and its role in development and pattern formation is one of the big challenges in biology today. Most attempts at tackling this problem either focus on the molecular details of transcription factor binding, or aim at genome-wide prediction of expression patterns from sequence through bioinformatics and mathematical modelling. Here we bridge the gap between these two complementary approaches by providing an integrative model of cis-regulatory elements governing the expression of the gap gene giant (gt) in the blastoderm embryo of Drosophila melanogaster. We use a reverse-engineering method, where mathematical models are fit to quantitative spatio-temporal reporter gene expression data to infer the regulatory mechanisms underlying gt expression in its anterior and posterior domains. These models are validated through prediction of gene expression in mutant backgrounds. A detailed analysis of our data and models reveals that gt is regulated by domain-specific CREs at early stages, while a late element drives expression in both the anterior and the posterior domains. Initial gt expression depends exclusively on inputs from maternal factors. Later, gap gene cross-repression and gt auto-activation become increasingly important. We show that auto-regulation creates a positive feedback, which mediates the transition from early to late stages of regulation. We confirm the existence and role of gt auto-activation through targeted mutagenesis of Gt transcription factor binding sites. In summary, our analysis provides a comprehensive picture of spatio-temporal gene regulation by different interacting enhancer elements for an important developmental regulator.A.H. received funding from the La Caixa Foundation to conduct her PhD project at the CRG. The laboratory of J.J. is funded by the MEC-EMBL agreement for the EMBL/CRG Research Unit in Systems Biology. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement FP7-KBBE-2011-5/289434 (BioPreDyn), by Grant 153 (MOPDEV) of the ERANet: ComplexityNET programme, by AGAUR SGR Grant 406, as well as grants BFU2009-10184 and BFU2012-33775 from the Spanish Ministry of the Economy and Competitiveness (MINECO, formerly MICINN). The Centre for Genomic Regulation (CRG) acknowledges support from MINECO, 'Centro de Excelencia Severo Ochoa 2013-2017', SEV-2012-0208

Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity

Abstract Background The development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive. Methods The SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed. Results A detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector. Conclusion A robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes

Cell-Autonomous Control of Neuronal Dendrite Expansion via the Fatty Acid Synthesis Regulator SREBP

Summary: During differentiation, neurons require a high lipid supply for membrane formation as they elaborate complex dendritic morphologies. While glia-derived lipids support neuronal growth during development, the importance of cell-autonomous lipid production for dendrite formation has been unclear. Using Drosophila larva dendritic arborization (da) neurons, we show that dendrite expansion relies on cell-autonomous fatty acid production. The nociceptive class four (CIV) da neurons form particularly large space-filling dendrites. We show that dendrite formation in these CIVda neurons additionally requires functional sterol regulatory element binding protein (SREBP), a crucial regulator of fatty acid production. The dendrite simplification in srebp mutant CIVda neurons is accompanied by hypersensitivity of srebp mutant larvae to noxious stimuli. Taken together, our work reveals that cell-autonomous fatty acid production is required for proper dendritic development and establishes the role of SREBP in complex neurons for dendrite elaboration and function. : Ziegler et al. highlight the endogenous role of fatty acid synthesis for proper neuronal dendrite growth during development. Using Drosophila da neurons, they show that large CIVda neurons cell-autonomously rely on fatty acid synthesis through the lipid synthesis master regulator SREBP. Keywords: Drosophila, dendrite differentiation, fatty acids, lipids, SREBP, metabolism, brain, nociceptio

Integral gene drives for population replacement

A first generation of CRISPR-based gene drives has now been tested in the laboratory in a number of organisms, including malaria vector mosquitoes. Challenges for their use in the area-wide genetic control of vector-borne disease have been identified, including the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. We have evaluated theoretically the concept of integral gene drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including drive and anti-pathogen effector elements directly embedded within endogenous genes – an arrangement that in theory allows targeting functionally conserved coding sequences without disrupting their function. Autonomous and non-autonomous IGD strains could be generated, optimized, regulated and imported independently. We performed quantitative modeling comparing IGDs with classical replacement drives and show that selection for the function of the hijacked host gene can significantly reduce the establishment of resistant alleles in the population, while drive occurring at multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target site variation. IGD thus has potential as a more durable and flexible population replacement strategy

Parameter correlations.

<p>This figure shows correlation matrices for parameter values derived from linear analysis (A), and bootstrapping (B), for <i>Kr</i> (green frame), <i>kni</i> (red frame), and <i>gt</i> (blue frame). Parameter notation: (production rate), (decay rate), (diffusion rate), and (production delay; see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003281#pcbi.1003281.e004" target="_blank">equation 1</a>). Colors indicate sign and strength of correlations. Matrices in (A) are calculated from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003281#pcbi.1003281.e131" target="_blank">equation 6</a> (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003281#s4" target="_blank">Materials and Methods</a>). Matrices in (B) are derived from the singular value decomposition of bootstrap distributions.</p

Comparison of model output and measured protein concentrations.

<p>(A) Spatial profiles of <i>Kr</i> (green), <i>kni</i> (red), and <i>gt</i> (blue) for early (T1), mid (T4), and late (T8) time classes during C14A. X-axes represent A–P position (in %), Y-axis show relative concentrations (as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003281#pcbi-1003281-g002" target="_blank">Figure 2A</a>). (B) Temporal dynamics of peak concentrations for the central <i>Kr</i> domain (left), the abdominal <i>kni</i> domain (centre), and the posterior <i>gt</i> domain (right). X-axes represent time, Y-axes show relative concentrations (as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003281#pcbi-1003281-g002" target="_blank">Figure 2C</a>). In all panels, model output is shown as a dashed black line; measured protein concentrations are shown as dark colored lines (mean) and lightly shaded background (standard deviations).</p

Confidence intervals for parameter estimates.

<p>This figure shows 95% confidence intervals for parameters (production rate), (decay rate), (diffusion rate), and (production delay; see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003281#pcbi.1003281.e004" target="_blank">equation 1</a>) for <i>Kr</i> (green), <i>kni</i> (red), and <i>gt</i> (blue). are independent, dependent intervals obtained from linear analysis (connected solid lines), are intervals obtained from bootstrapping (dashed lines). Dots (on solid lines) represent eSS parameter estimates, diamonds (on dashed lines) those from SA. Striped grey background indicates parameter values that lie outside the search space limits used for optimisation. Note that only a subregion of the search space is shown in each panel (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003281#s4" target="_blank">Materials and Methods</a> for values of search space limits).</p