High-resolution in situ analysis of Cas9 germline transcript distributions in gene-drive Anopheles mosquitoes

Abstract Gene drives are programmable genetic elements that can spread beneficial traits into wild populations to aid in vector-borne pathogen control. Two different drives have been developed for population modification of mosquito vectors. The Reckh drive (vasa-Cas9) in Anopheles stephensi displays efficient allelic conversion through males but generates frequent drive-resistant mutant alleles when passed through females. In contrast, the AgNos-Cd1 drive (nos-Cas9) in An. gambiae achieves almost complete allelic conversion through both genders. Here, we examined the subcellular localization of RNA transcripts in the mosquito germline. In both transgenic lines, Cas9 is strictly co-expressed with endogenous genes in stem and pre-meiotic cells of the testes, where both drives display highly efficient conversion. However, we observed distinct co-localization patterns for the two drives in female reproductive tissues. These studies suggest potential determinants underlying efficient drive through the female germline. We also evaluated expression patterns of alternative germline genes for future gene-drive designs.

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High throughput generation of promoter reporter (GFP) transgenic lines of low expressing genes in Arabidopsis and analysis of their expression patterns

Plant Methods ◽

10.1186/1746-4811-6-18 ◽

2010 ◽

Vol 6 (1) ◽

pp. 18 ◽

Cited By ~ 17

Author(s):

Yong-Li Xiao ◽

Julia C Redman ◽

Erin L Monaghan ◽

Jun Zhuang ◽

Beverly A Underwood ◽

...

Keyword(s):

High Throughput ◽

Expression Patterns ◽

Transgenic Lines

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Overexpression of a Banana Aquaporin Gene MaPIP1;1 Enhances Tolerance to Multiple Abiotic Stresses in Transgenic Banana and Analysis of Its Interacting Transcription Factors

Frontiers in Plant Science ◽

10.3389/fpls.2021.699230 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yi Xu ◽

Juhua Liu ◽

Caihong Jia ◽

Wei Hu ◽

Shun Song ◽

...

Keyword(s):

Abiotic Stresses ◽

Promoter Region ◽

Expression Patterns ◽

Soluble Sugar ◽

Ion Leakage ◽

Aquaporin Gene ◽

Gene Response ◽

Transcription Factor Genes ◽

Transgenic Lines ◽

Response To Stress

Aquaporins can improve the ability of plants to resist abiotic stresses, but the mechanism is still not completely clear. In this research, overexpression of MaPIP1;1 in banana improved tolerance to multiple stresses. The transgenic plants resulted in lower ion leakage and malondialdehyde content, while the proline, chlorophyll, soluble sugar, and abscisic acid (ABA) contents were higher. In addition, under high salt and recovery conditions, the content of Na+ and K+ is higher, also under recovery conditions, the ratio of K+/Na+ is higher. Finally, under stress conditions, the expression levels of ABA biosynthesis and response genes in the transgenic lines are higher than those of the wild type. In previous studies, we proved that the MaMADS3 could bind to the promoter region of MaPIP1;1, thereby regulating the expression of MaPIP1;1 and affecting the drought tolerance of banana plants. However, the mechanism of MaPIP1;1 gene response to stress under different adversity conditions might be regulated differently. In this study, we proved that some transcription factor genes, including MaERF14, MaDREB1G, MaMYB1R1, MaERF1/39, MabZIP53, and MaMYB22, showed similar expression patterns with MaPIP1;1 under salt or cold stresses, and their encoded proteins could bind to the promoter region of MaPIP1;1. Here we proposed a novel MaPIP1;1-mediated mechanism that enhanced salt and cold tolerance in bananas. The results of this study have enriched the stress-resistant regulatory network of aquaporins genes and are of great significance for the development of molecular breeding strategies for stress-resistant fruit crops.

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Transcription of Drosophila Troponin I Gene Is Regulated by Two Conserved, Functionally Identical, Synergistic Elements

Molecular Biology of the Cell ◽

10.1091/mbc.e03-09-0663 ◽

2004 ◽

Vol 15 (3) ◽

pp. 1185-1196 ◽

Cited By ~ 34

Author(s):

María-Cruz Marín ◽

José-Rodrigo Rodríguez ◽

Alberto Ferrús

Keyword(s):

Transcription Initiation ◽

Troponin I ◽

Regulatory Element ◽

Expression Patterns ◽

In Silico Analysis ◽

Initiation Site ◽

Transcription Initiation Site ◽

Upstream Regulatory Element ◽

Transgenic Lines

The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, we show that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects.

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Integral Gene Drives: an “operating system” for population replacement

10.1101/356998 ◽

2018 ◽

Cited By ~ 1

Author(s):

Alexander Nash ◽

Giulia Mignini Urdaneta ◽

Andrea K. Beaghton ◽

Astrid Hoermann ◽

Philippos Aris Papathanos ◽

...

Keyword(s):

Field Testing ◽

Target Site ◽

Gene Drive ◽

Population Replacement ◽

Pathogen Effector ◽

Site Variation ◽

The Face ◽

Endogenous Genes ◽

Target Site Resistance ◽

Gene Drives

AbstractFirst generation CRISPR-based gene drives have now been tested in the laboratory in a number of organisms including malaria vector mosquitoes. A number of challenges for their use in the area-wide genetic control of vector-borne disease have been identified. These include the development of target site resistance, their long-term efficacy in the field, their molecular complexity, and the practical and legal limitations for field testing of both gene drive and coupled anti-pathogen traits. To address these challenges, we have evaluated the concept of Integral Gene Drive (IGD) as an alternative paradigm for population replacement. IGDs incorporate a minimal set of molecular components, including both the drive and the anti-pathogen effector elements directly embedded within endogenous genes – an arrangement which we refer to as gene “hijacking”. This design would allow autonomous and non-autonomous IGD traits and strains to be generated, tested, optimized, regulated and imported independently. We performed quantitative modelling 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 hedging drive over multiple genomic loci prolongs the duration of transmission blockage in the face of pre-existing target-site variation. IGD thus has the potential to yield more durable and flexible population replacement traits.

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Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement

eLife ◽

10.7554/elife.58791 ◽

2021 ◽

Vol 10 ◽

Author(s):

Astrid Hoermann ◽

Sofia Tapanelli ◽

Paolo Capriotti ◽

Giuseppe Del Corsano ◽

Ellen KG Masters ◽

...

Keyword(s):

Regulatory Sequences ◽

Gene Drive ◽

Passive Mode ◽

Population Replacement ◽

Guide Rnas ◽

Efficient Gene ◽

Genetic Modifications ◽

Form Part ◽

Endogenous Genes ◽

Gene Drives

Gene drives for mosquito population replacement are promising tools for malaria control. However, there is currently no clear pathway for safely testing such tools in endemic countries. The lack of well-characterized promoters for infection-relevant tissues and regulatory hurdles are further obstacles for their design and use. Here we explore how minimal genetic modifications of endogenous mosquito genes can convert them directly into non-autonomous gene drives without disrupting their expression. We co-opted the native regulatory sequences of three midgut-specific loci of the malaria vector Anopheles gambiae to host a prototypical antimalarial molecule and guide-RNAs encoded within artificial introns that support efficient gene drive. We assess the propensity of these modifications to interfere with the development of Plasmodium falciparum and their effect on fitness. Because of their inherent simplicity and passive mode of drive such traits could form part of an acceptable testing pathway of gene drives for malaria eradication.

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High-throughput single-cell transcriptomics reveals the female germline differentiation trajectory in Arabidopsis thaliana

Communications Biology ◽

10.1038/s42003-021-02676-z ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Zhimin Hou ◽

Yanhui Liu ◽

Man Zhang ◽

Lihua Zhao ◽

Xingyue Jin ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Single Cell ◽

Cell Fate ◽

Single Cells ◽

Expression Patterns ◽

Cell Types ◽

Developmental Time ◽

Developmental Trajectory ◽

Germline Differentiation ◽

Female Germline

AbstractFemale germline cells in flowering plants differentiate from somatic cells to produce specialized reproductive organs, called ovules, embedded deep inside the flowers. We investigated the molecular basis of this distinctive developmental program by performing single-cell RNA sequencing (scRNA-seq) of 16,872 single cells of Arabidopsis thaliana ovule primordia at three developmental time points during female germline differentiation. This allowed us to identify the characteristic expression patterns of the main cell types, including the female germline and its surrounding nucellus. We then reconstructed the continuous trajectory of female germline differentiation and observed dynamic waves of gene expression along the developmental trajectory. A focused analysis revealed transcriptional cascades and identified key transcriptional factors that showed distinct expression patterns along the germline differentiation trajectory. Our study provides a valuable reference dataset of the transcriptional process during female germline differentiation at single-cell resolution, shedding light on the mechanisms underlying germline cell fate determination.

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Novel genome assemblies and Evolutionary Dynamics of North American Anopheles mosquitoes

10.1101/2021.08.31.458407 ◽

2021 ◽

Author(s):

Cory A Henderson ◽

Karen Kemirembe ◽

Sage Mckeand ◽

Christina M Bergey ◽

Jason L Rasgon

Keyword(s):

North American ◽

Evolutionary Dynamics ◽

Mosquito Species ◽

Evolutionary Relationship ◽

Single Copy ◽

Biological Traits ◽

Gene Drive ◽

Anopheles Mosquitoes ◽

The North ◽

Genome Assemblies

Anopheles mosquitoes are the principal vectors for malaria and lymphatic filariasis, and evidence for arboviral transmission under laboratory and natural contexts has been demonstrated. Vector management approaches require an understanding of the ecological, epidemiological, and biological context of the species in question, and increased interest in gene drive systems for vector control applications has resulted in an increased need for genome assemblies from understudied mosquito species. While the genomes for many Anopheles species have been sequenced, North American Anopheles taxa have been neglected. In this study we present novel genome assemblies for the North American species An. crucians, An. freeborni, An. albimanus, and An. quadrimaculatus, and examine the evolutionary relationship between these species. We identified 790 shared single copy orthologs between the newly sequenced genomes and created a phylogeny using 673 of the orthologs, identifying 525 orthologs with evidence for positive selection on at least one branch of the phylogeny. Gene ontology terms such as calcium ion signaling, histone binding, and protein acetylation were identified as being biased in the set of selected genes. These novel genome sequences will be useful in developing our understanding of the diverse biological traits that drive vectorial capacity in anophelines.

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Identification and characterization of a novel multi-stress responsive gene in Arabidopsis

PLoS ONE ◽

10.1371/journal.pone.0244030 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0244030

Author(s):

Faiza Tawab ◽

Iqbal Munir ◽

Zeeshan Nasim ◽

Mohammad Sayyar Khan ◽

Saleha Tawab ◽

...

Keyword(s):

Expression Patterns ◽

Survival Rates ◽

Crop Productivity ◽

Cell Wall Protein ◽

Transcriptome Data ◽

Gus Assay ◽

Transgenic Lines ◽

Responsive Gene ◽

Identification And Characterization

Abiotic stresses especially salinity, drought and high temperature result in considerable reduction of crop productivity. In this study, we identified AT4G18280 annotated as a glycine-rich cell wall protein-like (hereafter refer to as GRPL1) protein as a potential multistress-responsive gene. Analysis of public transcriptome data and GUS assay of pGRPL1::GUS showed a strong induction of GRPL1 under drought, salinity and heat stresses. Transgenic plants overexpressing GRPL1-3HA showed significantly higher germination, root elongation and survival rate under salt stress. Moreover, the 35S::GRPL1-3HA transgenic lines also showed higher survival rates under drought and heat stresses. GRPL1 showed similar expression patterns with Abscisic acid (ABA)-pathway genes under different growth and stress conditions, suggesting a possibility that GRPL1 might act in the ABA pathway that is further supported by the inability of ABA-deficient mutant (aba2-1) to induce GRPL1 under drought stress. Taken together, our data presents GRPL1 as a potential multi-stress responsive gene working downstream of ABA.

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Genetic reagents for making split-GAL4 lines in Drosophila

10.1101/197509 ◽

2017 ◽

Cited By ~ 3

Author(s):

Heather Dionne ◽

Karen L. Hibbard ◽

Amanda Cavallaro ◽

Jui-Chun Kao ◽

Gerald M. Rubin

Keyword(s):

Nervous System ◽

Genomic Dna ◽

Expression Patterns ◽

Cell Types ◽

Biological Processes ◽

Single Segment ◽

Distinct Cell ◽

Experimental Tool ◽

Transgenic Lines

AbstractThe ability to reproducibly target expression of transgenes to small, defined subsets of cells is a key experimental tool for understanding many biological processes. The Drosophila nervous system contains thousands of distinct cell types and it has generally not been possible to limit expression to one or a few cell types when using a single segment of genomic DNA as an enhancer to drive expression. Intersectional methods, in which expression of the transgene only occurs where two different enhancers overlap in their expression patterns, can be used to achieve the desired specificity. This report describes a set of over 2,800 transgenic lines for use with the split-GAL4 intersectional method.

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Genome-Wide Identification and Analysis on YUCCA Gene Family in Isatis indigotica Fort. and IiYUCCA6-1 Functional Exploration

International Journal of Molecular Sciences ◽

10.3390/ijms21062188 ◽

2020 ◽

Vol 21 (6) ◽

pp. 2188

Author(s):

Miaomiao Qin ◽

Jing Wang ◽

Tianyi Zhang ◽

Xiangyang Hu ◽

Rui Liu ◽

...

Keyword(s):

Gene Family ◽

Expression Patterns ◽

Control Group ◽

Binding Motif ◽

Isatis Indigotica ◽

Rate Limiting Step ◽

Genome Wide ◽

Transgenic Lines ◽

Gene Structures ◽

High Auxin

Auxin is one of the most critical hormones in plants. YUCCA (Tryptophan aminotransferase of Arabidopsis (TAA)/YUCCA) enzymes catalyze the key rate-limiting step of the tryptophan-dependent auxin biosynthesis pathway, from IPA (Indole-3-pyruvateacid) to IAA (Indole-3-acetic acid). Here, 13 YUCCA family genes were identified from Isatis indigotica, which were divided into four categories, distributing randomly on chromosomes (2n = 14). The typical and conservative motifs, including the flavin adenine dinucleotide (FAD)-binding motif and flavin-containing monooxygenases (FMO)-identifying sequence, existed in the gene structures. IiYUCCA genes were expressed differently in different organs (roots, stems, leaves, buds, flowers, and siliques) and developmental periods (7, 21, 60, and 150 days after germination). Taking IiYUCCA6-1 as an example, the YUCCA genes functions were discussed. The results showed that IiYUCCA6-1 was sensitive to PEG (polyethylene glycol), cold, wounding, and NaCl treatments. The over-expressed tobacco plants exhibited high auxin performances, and some early auxin response genes (NbIAA8, NbIAA16, NbGH3.1, and NbGH3.6) were upregulated with increased IAA content. In the dark, the contents of total chlorophyll and hydrogen peroxide in the transgenic lines were significantly lower than in the control group, with NbSAG12 downregulated and some delayed leaf senescence characteristics, which delayed the senescence process to a certain extent. The findings provide comprehensive insight into the phylogenetic relationships, chromosomal distributions, and expression patterns and functions of the YUCCA gene family in I. indigotica.

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