SYNCAS: Efficient CRISPR/Cas9 gene-editing in difficult to transform arthropods

Insect Biochemistry and Molecular Biology, Journal Year: 2024, Volume and Issue: 165, P. 104068 - 104068

Published: Jan. 1, 2024

The genome editing technique CRISPR/Cas9 has led to major advancements in many research fields and this state-of-the-art tool proven its use genetic studies for various arthropods. However, most transformation protocols rely on microinjection of component into embryos, a method which is challenging species. Alternatively, injections can be performed adult females, but efficiencies very low as was shown the two-spotted spider mite, Tetranychus urticae, minute important chelicerate pest crops. In study, we explored different formulations optimize maternal injection protocol T. urticae. We observed strong synergy between branched amphipathic peptide capsules saponins, resulting significant increase knock-out efficiency, exceeding 20%. This formulation, termed SYNCAS, used urticae genes – phytoene desaturase, CYP384A1 Antennapedia also allowed develop co-CRISPR strategy facilitated generation knock-in mutants. addition, SYNCAS successfully applied white white-like western flower thrips, Frankliniella occidentalis. allows routine these species game changer other hard transform

Language: Английский

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Model and Non-model Insects in Chronobiology

Frontiers in Behavioral Neuroscience, Journal Year: 2020, Volume and Issue: 14

Published: Nov. 26, 2020

The fruit fly Drosophila melanogaster is an established model organism in chronobiology, because genetic manipulation and breeding the laboratory are easy. circadian clock neuroanatomy D. one of best-known networks insects basic behavior has been characterized detail this insect. Another chronobiology honey bee Apis mellifera , which diurnal foraging described already early twentieth century. A. hallmarks research on interplay between sociality complex behaviors like sun compass navigation time-place-learning. Nevertheless, there aspects structure function, for example role photoperiodism diapause, can be only insufficiently investigated these two models. Unlike high-latitude flies such as Chymomyza costata or ezoana cosmopolitan do not display a photoperiodic diapause. Similarly, bees go into “real” but most solitary species exhibit obligatory Furthermore, evolved different Hymenoptera independently, wherefore it might misleading to study social Consequently, additional non-model required understand Diptera Hymenoptera. In review, we introduce compare them with other show their advantages limitations general models insect clocks.

Language: Английский

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Cas9-mediated gene editing in the black-legged tick, Ixodes scapularis, by embryo injection and ReMOT Control

iScience, Journal Year: 2022, Volume and Issue: 25(3), P. 103781 - 103781

Published: Feb. 15, 2022

Despite their capacity to acquire and pass on an array of debilitating pathogens, research ticks has lagged behind other arthropod vectors, such as mosquitoes, largely because challenges in applying available genetic molecular tools. CRISPR-Cas9 is transforming non-model organism research; however, successful gene editing not yet been reported ticks. Technical for injecting tick embryos attempt have further slowed progress. Currently, no embryo injection protocol exists any chelicerate species, including Herein, we report a the black-legged tick,

Language: Английский

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Targeted mutagenesis using CRISPR-Cas9 in the chelicerate herbivore Tetranychus urticae

Insect Biochemistry and Molecular Biology, Journal Year: 2020, Volume and Issue: 120, P. 103347 - 103347

Published: Feb. 27, 2020

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and insect species. However, successful gene editing or transformation not yet been reported for chelicerates, the second largest group terrestrial animals. Within this group, some mite tick species are economically very important agriculture human health, availability a gene-editing tool would be significant advancement field. Here, we report on spider Tetranychus urticae. ovary virgin adult females was injected with mix Cas9 sgRNAs targeting phytoene desaturase gene. Natural mutants laterally transferred have previously shown an easy-to-score albino phenotype. Albino sons were mated wild-type females, two independently transformed lines created further characterized. Albinism inherited as recessive monogenic trait. Sequencing complete target-gene both revealed different lesions at expected locations near PAM site target-gene. Both did genetically complement each other dedicated crosses, nor when crossed to reference strain known defect same In conclusion, independent mutagenesis events induced T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that can used create knockouts mites.

Language: Английский

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Pterin-based pigmentation in animals

Biology Letters, Journal Year: 2021, Volume and Issue: 17(8), P. 20210221 - 20210221

Published: Aug. 1, 2021

Pterins are one of the major sources bright coloration in animals. They produced endogenously, participate vital physiological processes and serve a variety signalling functions. Despite their ubiquity nature, pterin-based pigmentation has received little attention when compared to other pigment classes. Here, we summarize aspects relating pterin animals, from its long history research recent genomic studies on molecular mechanisms underlying evolution. We argue that pterins have intermediate characteristics (endogenously produced, typically bright) between two well-studied types, melanins cryptic) carotenoids (dietary uptake, bright), providing unique opportunities address general questions about biology coloration, determine how different types evolve discussions honest hypotheses. Crucial gaps persist our knowledge basis production deposition pterins. thus highlight need for functional systems amenable laboratory manipulation, but also exhibit natural variation pigmentation. The wealth potential model species, coupled with technological analytical advances, make this promising time advance

Language: Английский

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Loss of Timeless Underlies an Evolutionary Transition within the Circadian Clock

Molecular Biology and Evolution, Journal Year: 2021, Volume and Issue: 39(1)

Published: Dec. 2, 2021

Abstract Most organisms possess time-keeping devices called circadian clocks. At the molecular level, clocks consist of transcription–translation feedback loops (TTFLs). Although some components negative TTFL are conserved across animals, important differences exist between typical models, such as mouse and fruit fly. In Drosophila, key PERIOD (PER) TIMELESS (TIM-d) proteins, whereas mammalian clock relies on PER CRYPTOCHROME (CRY-m). Importantly, how has maintained functionality during evolutionary transitions different states remains elusive. Therefore, we systematically described gene setup in major bilaterian lineages identified marked lineage-specific their constitution. Then performed a thorough functional analysis linden bug Pyrrhocoris apterus, an insect species comprising features characteristic both Drosophila Unexpectedly, knockout timeless-d, essential for ticking did not compromise rhythmicity P. it only accelerated its pace. Furthermore, silencing timeless-m, ancestral timeless type ubiquitously present resulted mild gradual loss rhythmicity, supporting possible participation clock, which is consistent with timeless-m role suggested by research models. The dispensability timeless-d apterus allows drawing scenario remained at each step transition from state to TIM-d-independent + CRY-m system operating extant vertebrates, including humans.

Language: Английский

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Pigment Dispersing Factor Is a Circadian Clock Output and Regulates Photoperiodic Response in the Linden Bug, Pyrrhocoris apterus

Frontiers in Physiology, Journal Year: 2022, Volume and Issue: 13

Published: April 29, 2022

Daily and annually cycling conditions manifested on the Earth have forced organisms to develop time-measuring devices. Circadian clocks are responsible for adjusting physiology daily cycles in environment, while anticipation of seasonal changes is governed by photoperiodic clock. cell-autonomous depend transcriptional/translational feedback loops conserved clock genes. The synchronization among centers brain achieved modulatory function clock-dependent neuropeptides. In insects, most prominent neuropeptide Pigment Dispersing Factor (PDF). Photoperiodic measures computes day and/or night length adjusts accordingly upcoming season. exact mechanism its direct signaling molecules unknown but, many circadian genes involved responses. While Drosophila, PDF participates both output diapause regulation, weak response curve D. melanogaster a major limitation revealing full role Here we provide first description linden bug, Pyrrhocoris apterus, an organism with robust response. We characterize detail phenotype several CRISPR/Cas9-generated pdf mutants, including three null mutants two modified PDF. Our results show that acts downstream CRY plays key as output. Surprisingly, contrast diurnal activity wild-type bugs, predominantly nocturnal activity, which caused clock-independent light/dark switch. Moreover, together CRY, photoperiod-dependent induction, however, lack does not disrupt completely, suggesting presence additional clock-regulated factors. Taken our data new insight into insect's systems.

Language: Английский

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Functional genomic tools for emerging model species

Trends in Ecology & Evolution, Journal Year: 2022, Volume and Issue: 37(12), P. 1104 - 1115

Published: July 30, 2022

Understanding the molecular mechanisms underlying phenotypic evolution is a central goal in evolutionary biology.Unfortunately, causal associations between candidate loci and phenotype are rarely established because majority of never validated with functional tools.We describe latest developments use tools, particularly RNAi clustered regularly interspaced palindromic repeats (CRISPR)-Cas, to establish links genotype phenotype, discuss some methodological challenges when applying these tools that unique emerging model organisms.Specifically, we highlight significant issue current studies ecology biology date recapitulate ecologically relevant variation, suggest gene editing species should make more HDR pathway obtain insights thereby deeper understanding genetic basis traits. Most field aiming connect validate identified using tools. Recent RNA interference (RNAi) (CRISPR)-Cas genome have dramatically increased feasibility validation. However, methods come specific applied organisms, including limited spatial control silencing, low knock-in efficiencies, throughput Moreover, many do not this limits their scope for into processes. We therefore argue by allelic replacement through homology-directed repair (HDR) would greatly benefit evolution. A fundamental aim identify genomic variation underlies micro- macroevolutionary dynamics [1.Vitti J.J. et al.Detecting natural selection data.Annu. Rev. Genet. 2013; 47: 97-120Crossref PubMed Scopus (397) Google Scholar]. In present post-genomic era, diverse datasets routinely used purpose organisms (see Glossary), ranging from genome-wide population data various association approaches differential expression analysis chromatin accessibility assays [2.Jamann T.M. al.QTL mapping high-throughput sequencing.in: Alonso J.M. Stepanova A.N. Plant Functional Genomics: Methods Protocols. Springer, 2015: 257-285Crossref (20) Scholar,3.van der Burg K.R.L. al.Genomic architecture genetically assimilated seasonal color pattern.Science. 2020; 370: 721-725Crossref (24) Although such advancing our genotype–phenotype relationships traits involved adaptation speciation [3.van Scholar,4.Pardo-Diaz C. al.Towards identification adaptive evolution.Methods Ecol. Evol. 2015; 6: 445-464Crossref (93) Scholar], they also clear limitations, most obvious being lack ability rigorous inferences phenotypes genes or loci. manipulating function, as (CRISPR)–CRISPR-associated protein (Cas), recent years made it possible link organisms. review address growing need inferences, practical aspects validation future role CRISPR-Cas technologies evolution, strong emphasis on challenge importance establishing ecological relevance Rather than investing validation, genomics hasinstead predominantly resulted an increase sample sizes individuals, populations, quest speciation. The result ever-increasing number that, after identifying (e.g., tests selection, allele-frequency patterns, mapping, linkage analysis, transcriptomic studies), at length potential implications. These all inherent (reviewed [5.Schielzeth H. al.Success failure replication associations: How does help outbred populations?.Mol. Resour. 2018; 18: 739-754Crossref (19) Scholar, 6.Liu Y. al.On dependency cellular levels mRNA abundance.Cell. 2016; 165: 535-550Abstract Full Text PDF (1576) 7.Schielzeth Husby A. Challenges prospects quantitative trait standing populations.Ann. N. Acad. Sci. 2014; 1320: 35-57Crossref (41) 8.Wellenreuther M. Hansson B. Detecting polygenic evolution: problems, pitfalls, promises.Trends TIG. 32: 155-164Abstract (84) Scholar]), but common them limitation phenotype. Drawing conclusions body evidence challenging, interpretations be considered carefully attempting understand relationship. Given circumstances, raise two worth considering investigating First, although acknowledge implementation even desirable instances, wish stress absence proper allowing avoiding language. Imprecise language degrades insights, impedes how world works, bloats false positive results [9.Siddiq M.A. al.Experimental test refutation classic case Drosophila melanogaster.Nat. 2017; 1: 25Crossref (21) Second, relying upon existing annotations without perpetuates bias. Consider hypothetical scenario, wherein intersection SNP trait, where variants associated near coding sequences several differentially expressed genes. resulting list assessed, hypotheses formed based sequence element annotations, which turn overwhelmingly closest organism melanogaster insects, mouse Mus musculus mammals, zebrafish Danio rerio fish, Saccharomyces cerevisiae yeast, Arabidopsis thaliana plants, so on). note duality approach: seems sound way forward homologous share functions over deep time, approach necessary no other possible, comes risk erroneous inferences. Model only important unknown function [10.Wu al.CRISPR/Cas9 mediated resource kinase phosphatase Drosophila.Sci. 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They then manipulate four Knockout (KO) first choice, locus annotated wing-pattern (cortex), predictably affected color. Of one had effect (Dscam3) whereas did (trehalase). Finally, KO uncharacterized unannotated demonstrated causally coloration plasticity, was named herfst. Had chosen cortex, novel missed, (the metabolic trehalase) has unpredicted patterning. conducted neuronal Dscam3 patterning might generated line inquiry fact lacks support, trehalase possibly discounted. discovery herfst provides knowledge allows reanalysis previous work. With components types will synergistically improve across broader taxonomical range interest field. Establishing necessity stepwise process requires determination effort. amenable manipulation, others accomplished scale months, led impressive proliferation insightful knockdown techniques explore candidates were previously association-based [14.Klaassen al.CRISPR mutagenesis confirms oca2 melanin pigmentation Astyanax mexicanus.Dev. 441: 313-318Crossref (59) 15.Li X. al.Outbred sequencing CRISPR/Cas9 butterflies.Nat. Commun. 8212Crossref (133) 16.Mazo-Vargas al.Macroevolutionary shifts WntA potentiate diversity.Proc. Natl. U. S. 114: 10701-10706Crossref (99) 17.Santos M.E. al.Taxon-restricted origin access new environment.Science. 358: 386-390Crossref (56) 18.Woronik al.A transposable insertion alternative life history strategy.Nat. 2019; 5757Crossref (28) 19.Wucherpfennig J.I. al.Efficient CRISPR-Cas9 major sticklebacks.Evol. Res. 20: 107-132PubMed 20.Zhang L. al.Single master regulatory coordinates development iridescence.Proc. 10707-10712Crossref (97) taxonomically diverse, confirming butterflies [15.Li Scholar,16.Mazo-Vargas Scholar,20.Zhang albinism cave fish describing habitats water striders [17.Santos findings benefits bringing realm possibility behind processes documented populations difficult include phenomena migration [21.Zhan al.The genetics monarch warning colouration.Nature. 514: 317-321Crossref (186) exaggeration [22.Toubiana W. Khila Fluctuating strength intense male competition underlie strider's weapon.Proc. 28620182400PubMed innovations [23.Wagner G.P. Lynch V.J. Evolutionary novelties.Curr. 2010; R48-R52Abstract (184) polyphenisms [24.Casasa al.Nutrition-responsive insect polyphenism.Nat. 4: 970-978Crossref (17) name few. technique artificially downregulate assess level. downregulation occurs temporarily usually referred reduces rather eliminates production, opposed KO. largely relies proteins endogenous small pathways microRNA, interfering RNA, Piwi-interacting RNA) eukaryotic lineages [25.Shabalina S.A. Koonin E.V. Origins interference.Trends 2008; 23: 578-587Abstract (364) taxa highly divergent complex [26.Chapman E.J. Carrington J.C. Specialization pathways.Nat. 2007; 884-896Crossref (557) exogenously induced follow simplified conserved mechanism (Box 1) makes (although there notable exceptions; e.g., Lepidoptera [27.Terenius O. al.RNA Lepidoptera: overview successful unsuccessful implications experimental design.J. 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Genomics. 2011; 238-247Crossref (12) Scholar].The dependent transport cytosol described above initiated. groups efficient, vertebrates, cell membranes occur easily. cases, introduction aided exogenous agents viral vectors electroporation, delivering vehicle cationic lipids microinjection, solution injected directly tissue. This method efficient transport, systemic RNAi. strategy can, large extent, guided nearest organism. Apart generally broad applicability taxa, advantages stages tissue localization, both minimize negative pleiotropic effects. Further, tuned controlling dose completely ablated [28.Killiny al.Double-stranded uptake topical application, five CYP4 suppresses insecticide resistance Diaphorina citri.PLoS One. 9e110536Crossref (116) Scholar,29.Refki P.N. al.Emergence sensitivity Hox morphological trait.Dev. 392: 441-453Crossref (27) thus enabling survival phenotyping infer function. 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Cell. 691-704Abstract (94) addition RNAi, application insects [39.Wang al.Second-generation supply effective targets pest control.PLoS 6e18644Google underutilized screens. One obstacle efficiency delivery within treated individual varies [40.Vogel insects: protecting beneficials pests.Front. 9: 1912Crossref (107) generating necessitates larger sizes. Another drawback lacking administration difficult. vertebrates resort via [41.Schepers Practice: Principles, Basics, Silencing elegans, Drosophila, Mammals. John Wiley & Sons,

Language: Английский

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Efficient CRISPR/Cas9-mediated genome modification of the glassy-winged sharpshooter Homalodisca vitripennis (Germar)

Scientific Reports, Journal Year: 2022, Volume and Issue: 12(1)

Published: April 19, 2022

Abstract CRISPR/Cas9 technology enables the extension of genetic techniques into insect pests previously refractory to analysis. We report establishment analysis in glassy-winged sharpshooter (GWSS), Homalodisca vitripennis , which is a significant leafhopper pest agriculture California. use novel and simple approach embryo microinjection situ on host plant obtain high frequency mutagenesis, excess 55%, cinnabar white eye pigmentation loci. Through pair matings, we obtained 100% transmission w cn alleles G3 generation also established that both genes are located autosomes. Our wing phenotype revealed an unexpected discovery participation pteridine pigments wing-vein coloration, indicating role for these beyond color. used amplicon sequencing examine extent off-target mutagenesis adults arising from injected eggs, was found be negligible or non-existent. data show GWSS can easily developed as model system Hemiptera, enabling study traits contribute success invasive vectors pathogens. This will facilitate control strategies.

Language: Английский

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Gene Editing and Genetic Control of Hemipteran Pests: Progress, Challenges and Perspectives

Frontiers in Bioengineering and Biotechnology, Journal Year: 2022, Volume and Issue: 10

Published: June 7, 2022

The origin of the order Hemiptera can be traced to late Permian Period more than 230 MYA, well before flowering plants 100 MY later in during Cretaceous period. Hemipteran species consume their liquid diets using a sucking proboscis; for phytophagous hemipterans mouthparts (stylets) are elegant structures that enable voracious feeding from plant xylem or phloem. This adaptation has resulted some hemipteran becoming globally significant pests agriculture resulting annual crop losses. Due reliance on chemical insecticides control insect agricultural settings, many have evolved resistance an urgent need develop new, species-specific and environmentally friendly methods pest control. rapid advances CRISPR/Cas9 technologies model insects such as Drosophila melanogaster , Tribolium castaneum Bombyx mori, Aedes aegypti spurred new round innovative genetic strategies Diptera Lepidoptera increased interest assessing Hemiptera. Genetic approaches have, date, been largely overlooked due problems introducing material into germline these insects. high frequency CRISPR-mediated mutagenesis suggest that, if delivery problem could solved, then gene editing might quickly achieved. Significant realized nine over past 4 years. Here we review progress discuss challenges opportunities extending contemporary this agriculturally important r.

Language: Английский

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