scholarly journals Evolution of Resistance Against CRISPR/Cas9 Gene Drive

2016 ◽  
Author(s):  
Robert L. Unckless ◽  
Andrew G. Clark ◽  
Philipp W. Messer
Keyword(s):  
De Novo ◽  
Natural Populations ◽  
Resistance Mechanisms ◽  
Target Site ◽  
Pest Species ◽  
Gene Drive ◽  
Guide Rna ◽  
Deleterious Alleles ◽  
Laboratory Populations ◽  
Potential Applications

AbstractThe idea of driving genetically modified alleles to fixation in a population has fascinated scientists for over 40 years1,2. Potential applications are broad and ambitious, including the eradication of disease vectors, the control of pest species, and the preservation of endangered species from extinction3. Until recently, these possibilities have remained largely abstract due to the lack of an effective drive mechanism. CRISPR/Cas9 gene drive (CGD) now promise a highly adaptable approach for driving even deleterious alleles to high population frequency, and this approach was recently shown to be effective in small laboratory populations of insects4–7. However, it remains unclear whether CGD will also work in large natural populations in the face of potential resistance mechanisms. Here we show that resistance against CGD will inevitably evolve unless populations are small and repair of CGD-induced cleavage via nonhomologous end joining (NHEJ) can be effectively suppressed, or resistance costs are on par with those of the driver. We specifically calculate the probability that resistance evolves from variants at the target site that are not recognized by the driver's guide RNA, either because they are already present when the driver allele is introduced, arise byde novomutation, or are created by the driver itself when NHEJ introduces mutations at the target site. Our results shed light on strategies that could facilitate the engineering of a successful drive by lowering resistance potential, as well as strategies that could promote resistance as a possible mechanism for controlling a drive. This study highlights the need for careful modeling of CGD prior to the actual release of a driver construct into the wild.

scholarly journals The mechanism of potato resistance to Globodera rostochiensis: comparison of root transcriptomes of resistant and susceptible Solanum phureja genotypes

2020 ◽  
Vol 20 (S1) ◽  
Author(s):  
Alex V. Kochetov ◽  
Anastasiya A. Egorova ◽  
Anastasiya Y. Glagoleva ◽  
Kseniya V. Strygina ◽  
Elena K. Khlestkina ◽  
...  
Keyword(s):  
De Novo ◽  
Expression Patterns ◽  
Durable Resistance ◽  
Natural Populations ◽  
Resistance Mechanisms ◽  
Globodera Rostochiensis ◽  
Solanum Phureja ◽  
R Genes ◽  
Molecular Events ◽  
High Level

Abstract Background Globodera rostochiensis belongs to major potato pathogens with a sophisticated mechanism of interaction with roots of the host plants. Resistance of commercial varieties is commonly based on specific R genes introgressed from natural populations of related wild species and from native potato varieties grown in the Andean highlands. Investigation of molecular resistance mechanisms and screening the natural populations for novel R genes are important for both fundamental knowledge on plant pathogen interactions and breeding for durable resistance. Here we exploited the Solanum phureja accessions collected in South America with contrasting resistance to G. rostochiensis. Results The infestation of S. phureja with G. rostochiensis juveniles resulted in wounding stress followed by activation of cell division and tissue regeneration processes. Unlike the susceptible S. phureja genotype, the resistant accession reacted by rapid induction of variety of stress response related genes. This chain of molecular events accompanies the hypersensitive response at the juveniles’ invasion sites and provides high-level resistance. Transcriptomic analysis also revealed considerable differences between the analyzed S. phureja genotypes and the reference genome. Conclusion The molecular processes in plant roots associated with changes in gene expression patterns in response to G. rostochiensis infestation and establishment of either resistant or susceptible phenotypes are discussed. De novo transcriptome assembling is considered as an important tool for discovery of novel resistance traits in S. phureja accessions.

scholarly journals Dodging silver bullets: good CRISPR gene-drive design is critical for eradicating exotic vertebrates

2017 ◽  
Vol 284 (1860) ◽  
pp. 20170799 ◽  
Author(s):  
Thomas A. A. Prowse ◽  
Phillip Cassey ◽  
Joshua V. Ross ◽  
Chandran Pfitzner ◽  
Talia A. Wittmann ◽  
...  
Keyword(s):  
Female Sterility ◽  
Gene Drive ◽  
End Joining ◽  
Guide Rna ◽  
Deleterious Alleles ◽  
Precise Knowledge ◽  
Animal Populations ◽  
Individual Based Models ◽  
Non Homologous End Joining ◽  
Gene Drives

Self-replicating gene drives that can spread deleterious alleles through animal populations have been promoted as a much needed but controversial ‘silver bullet’ for controlling invasive alien species. Homing-based drives comprise an endonuclease and a guide RNA (gRNA) that are replicated during meiosis via homologous recombination. However, their efficacy for controlling wild populations is threatened by inherent polymorphic resistance and the creation of resistance alleles via non-homologous end-joining (NHEJ)-mediated DNA repair. We used stochastic individual-based models to identify realistic gene-drive strategies capable of eradicating vertebrate pest populations (mice, rats and rabbits) on islands. One popular strategy, a sex-reversing drive that converts heterozygous females into sterile males, failed to spread and required the ongoing deployment of gene-drive carriers to achieve eradication. Under alternative strategies, multiplexed gRNAs could overcome inherent polymorphic resistance and were required for eradication success even when the probability of NHEJ was low. Strategies causing homozygotic embryonic non-viability or homozygotic female sterility produced high probabilities of eradication and were robust to NHEJ-mediated deletion of the DNA sequence between multiplexed endonuclease recognition sites. The latter two strategies also purged the gene drive when eradication failed, therefore posing lower long-term risk should animals escape beyond target islands. Multiplexing gRNAs will be necessary if this technology is to be useful for insular extirpation attempts; however, precise knowledge of homing rates will be required to design low-risk gene drives with high probabilities of eradication success.

scholarly journals Next-generation gene drive for population modification of the malaria vector mosquito, Anopheles gambiae

2020 ◽  
Vol 117 (37) ◽  
pp. 22805-22814 ◽  
Author(s):  
Rebeca Carballar-Lejarazú ◽  
Christian Ogaugwu ◽  
Taylor Tushar ◽  
Adam Kelsey ◽  
Thai Binh Pham ◽  
...  
Keyword(s):  
Anopheles Gambiae ◽  
Malaria Vector ◽  
Genetic Load ◽  
Target Site ◽  
Performance Criteria ◽  
Gene Drive ◽  
Guide Rna ◽  
Vector Mosquito

A Cas9/guide RNA-based gene drive strain, AgNosCd-1, was developed to deliver antiparasite effector molecules to the malaria vector mosquito, Anopheles gambiae. The drive system targets the cardinal gene ortholog producing a red-eye phenotype. Drive can achieve 98 to 100% in both sexes and full introduction was observed in small cage trials within 6 to 10 generations following a single release of gene-drive males. No genetic load resulting from the integrated transgenes impaired drive performance in the trials. Potential drive-resistant target-site alleles arise at a frequency <0.1, and five of the most prevalent polymorphisms in the guide RNA target site in collections of colonized and wild-derived African mosquitoes do not prevent cleavage in vitro by the Cas9/guide RNA complex. Only one predicted off-target site is cleavable in vitro, with negligible deletions observed in vivo. AgNosCd-1 meets key performance criteria of a target product profile and can be a valuable component of a field-ready strain for mosquito population modification to control malaria transmission.

scholarly journals De novo design of a reversible phosphorylation-dependent switch for membrane targeting

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Leon Harrington ◽  
Jordan M. Fletcher ◽  
Tamara Heermann ◽  
Derek N. Woolfson ◽  
Petra Schwille
Keyword(s):  
Protein Interactions ◽  
Lipid Membrane ◽  
De Novo ◽  
Protein Localization ◽  
Protein Structures ◽  
Spatiotemporal Pattern ◽  
Membrane Targeting ◽  
Protein Protein Interactions ◽  
Reversible Phosphorylation ◽  
Potential Applications

AbstractModules that switch protein-protein interactions on and off are essential to develop synthetic biology; for example, to construct orthogonal signaling pathways, to control artificial protein structures dynamically, and for protein localization in cells or protocells. In nature, the E. coli MinCDE system couples nucleotide-dependent switching of MinD dimerization to membrane targeting to trigger spatiotemporal pattern formation. Here we present a de novo peptide-based molecular switch that toggles reversibly between monomer and dimer in response to phosphorylation and dephosphorylation. In combination with other modules, we construct fusion proteins that couple switching to lipid-membrane targeting by: (i) tethering a ‘cargo’ molecule reversibly to a permanent membrane ‘anchor’; and (ii) creating a ‘membrane-avidity switch’ that mimics the MinD system but operates by reversible phosphorylation. These minimal, de novo molecular switches have potential applications for introducing dynamic processes into designed and engineered proteins to augment functions in living cells and add functionality to protocells.

scholarly journals Natural variation in yolk fatty acids, but not androgens, predicts offspring fitness in a wild bird

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Lucia Mentesana ◽  
Martin N. Andersson ◽  
Stefania Casagrande ◽  
Wolfgang Goymann ◽  
Caroline Isaksson ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Polyunsaturated Fatty Acids ◽  
Maternal Effects ◽  
Natural Variation ◽  
De Novo ◽  
Natural Populations ◽  
Egg Laying ◽  
Interactive Effects ◽  
Free Living ◽  
Offspring Fitness

Abstract Background In egg-laying animals, mothers can influence the developmental environment and thus the phenotype of their offspring by secreting various substances into the egg yolk. In birds, recent studies have demonstrated that different yolk substances can interactively affect offspring phenotype, but the implications of such effects for offspring fitness and phenotype in natural populations have remained unclear. We measured natural variation in the content of 31 yolk components known to shape offspring phenotypes including steroid hormones, antioxidants and fatty acids in eggs of free-living great tits (Parus major) during two breeding seasons. We tested for relationships between yolk component groupings and offspring fitness and phenotypes. Results Variation in hatchling and fledgling numbers was primarily explained by yolk fatty acids (including saturated, mono- and polyunsaturated fatty acids) - but not by androgen hormones and carotenoids, components previously considered to be major determinants of offspring phenotype. Fatty acids were also better predictors of variation in nestling oxidative status and size than androgens and carotenoids. Conclusions Our results suggest that fatty acids are important yolk substances that contribute to shaping offspring fitness and phenotype in free-living populations. Since polyunsaturated fatty acids cannot be produced de novo by the mother, but have to be obtained from the diet, these findings highlight potential mechanisms (e.g., weather, habitat quality, foraging ability) through which environmental variation may shape maternal effects and consequences for offspring. Our study represents an important first step towards unraveling interactive effects of multiple yolk substances on offspring fitness and phenotypes in free-living populations. It provides the basis for future experiments that will establish the pathways by which yolk components, singly and/or interactively, mediate maternal effects in natural populations.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

scholarly journals Comparative Study of the Effect of Solvents on the Efficacy of Neonicotinoid Insecticides Against Malaria Vector Populations Across Africa

2022 ◽  
Author(s):  
Magellan Tchouakui ◽  
Tatiane Assatse ◽  
Leon M. J. Mugenzi ◽  
Benjamin D. Menze ◽  
Daniel Nguiffo-Nguete ◽  
...  
Keyword(s):  
Democratic Republic Of Congo ◽  
Natural Populations ◽  
Resistance Mechanisms ◽  
Lower Mortality ◽  
Malaria Vectors ◽  
Cross Resistance ◽  
Metabolic Resistance ◽  
Resistance Development ◽  
Development Of Resistance ◽  
Bottle Test

Abstract Background New insecticides with a novel mode of action such as neonicotinoids have recently been recommended for public health by WHO. Resistance monitoring of such novel insecticides requires a robust protocol to monitor the development of resistance in natural populations. In this study, we comparatively used three different solvents to assess the susceptibility of malaria vectors to neonicotinoids across Africa.MethodsMosquitoes were collected from May to July 2021 from three agricultural settings in Cameroon (Njombe-Penja, Nkolondom, and Mangoum), the Democratic Republic of Congo (Ndjili-Brasserie), Ghana (Obuasi), and Uganda (Mayuge). Using the CDC bottle test, we compared the effect of three different solvents (ethanol, acetone, MERO) on the efficacy of neonicotinoids against Anopheles gambiae s.l. In addition, TaqMan assays were used to genotype key pyrethroid-resistant markers in An. gambiae and to evaluate potential cross-resistance between pyrethroids and clothianidin.ResultsLower mortality were observed when using absolute ethanol or acetone alone as solvent (11.4- 51.9% mortality in Nkolondom, 31.7- 48.2% in Mangoum, 34.6- 56.1% in Mayµge, 39.4- 45.6% in Obuasi, 83.7- 89.3% in Congo and 71.05- 95.9% in Njombe pendja) compared to acetone + MERO for which 100% mortality were observed for all the populations. Synergist assays (PBO, DEM and DEF) revealed a significant increase of mortality suggesting that metabolic resistance mechanisms are contributing to the reduced susceptibility. A negative association was observed between the L1014F-kdr mutation and clothianidin resistance with a greater frequency of homozygote resistant mosquitoes among the dead than among survivors (OR=0.5; P=0.02). However, the I114T-GSTe2 was in contrast significantly associated with a greater ability to survive clothianidin with a higher frequency of homozygote resistant among survivors than other genotypes (OR=2.10; P=0.013). ConclusionsThis study revealed a contrasted susceptibility pattern depending on the solvents with ethanol/acetone resulting to lower mortality, thus possibly overestimating resistance, whereas the MERO consistently showed a greater efficacy of neonicotinoids but it could prevent to detect early resistance development. Therefore, we recommend monitoring the susceptibility using both acetone alone and acetone+MERO (8-10µg/ml for clothianidin) to capture the accurate resistance profile of the mosquito populations.

scholarly journals Capturing the transcription factor interactome in response to sub-lethal insecticide exposure

2020 ◽  
Author(s):  
Victoria A Ingham ◽  
Sara Elg ◽  
Sanjay C Nagi ◽  
Frank Dondelinger
Keyword(s):  
Transcription Factors ◽  
Time Course ◽  
Lethal Dose ◽  
Resistance Mechanisms ◽  
Dynamic Bayesian Networks ◽  
Disease Vectors ◽  
Anopheles Coluzzii ◽  
Pest Species ◽  
Agricultural Pests ◽  
Insecticide Exposure

AbstractThe increasing levels of pesticide resistance in agricultural pests and disease vectors represents a threat to both food security and global health. As insecticide resistance intensity strengthens and spreads, the likelihood of a pest encountering a sub-lethal dose of pesticide dramatically increases. Here, we apply dynamic Bayesian networks to a transcriptome time-course generated using sub-lethal pyrethroid exposure on a highly resistant Anopheles coluzzii population. The model accounts for circadian rhythm and ageing effects allowing high confidence identification of transcription factors with key roles in pesticide response. The associations generated by this model show high concordance with lab-based validation and identifies 44 transcription factors regulating insecticide-responsive transcripts. We identify six key regulators, with each displaying differing enrichment terms, demonstrating the complexity of pesticide response. The considerable overlap of resistance mechanisms in agricultural pests and disease vectors strongly suggests that these findings are relevant in a wide variety of pest species.

scholarly journals Investigation of the influence of a glutathione S-transferase metabolic resistance to pyrethroids/DDT on mating competitiveness in males of the African malaria vector, Anopheles funestus

2019 ◽  
Vol 4 ◽  
pp. 13 ◽  
Author(s):  
Magellan Tchouakui ◽  
Billy Tene Fossog ◽  
Brigitte Vanessa Ngannang ◽  
Doumani Djonabaye ◽  
Williams Tchapga ◽  
...  
Keyword(s):  
Natural Populations ◽  
Anopheles Funestus ◽  
Resistance Mechanisms ◽  
Malaria Vectors ◽  
Glutathione S Transferase ◽  
Metabolic Resistance ◽  
Mating Competitiveness ◽  
Metabolic Marker ◽  
Susceptible Allele ◽  
Permanet 3.0

Background: Metabolic resistance is a serious challenge to current insecticide-based interventions. The extent to which it affects natural populations of mosquitoes including their reproduction ability remains uncharacterised. Here, we investigated the potential impact of the glutathione S-transferase L119F-GSTe2 resistance on the mating competitiveness of male Anopheles funestus, in Cameroon. Methods: Swarms and indoor resting collections took place in March, 2018 in Tibati, Cameroon. WHO tube and cone assays were performed on F1 mosquitoes from indoor collected females to assess the susceptibility profile of malaria vectors. Mosquitoes mated and unmated males collected in the swarms were genotyped for the L119F metabolic marker to assess its association with mating male competitiveness. Results: Susceptibility and synergist assays, showed that this population was multiple resistant to pyrethroids, DDT and carbamates, likely driven by metabolic resistance mechanisms. Cone assays revealed a reduced efficacy of standard pyrethroid-nets (Olyset and PermaNet 2.0) with low mortality (<25%) whereas synergist PBO-Nets (Olyset Plus and PermaNet 3.0) retained greater efficacy with higher mortality (>80%). The L119F-GSTe2 mutation, conferring pyrethroid/DDT resistance, was detected in this An. funestus population at a frequency of 28.8%. In addition, a total of 15 mating swarms were identified and 21 An. funestus couples were isolated from those swarms.  A comparative genotyping of the L119F-GSTe2 mutation between mated and unmated males revealed that heterozygote males 119L/F-RS were less able to mate than homozygote susceptible (OR=7.2, P<0.0001). Surprisingly, heterozygote mosquitoes were also less able to mate than homozygote resistant (OR=4.2, P=0.010) suggesting the presence of a heterozygote disadvantage effect. Overall, mosquitoes bearing the L119-S susceptible allele were significantly more able to mate than those with 119F-R resistant allele (OR=2.1, P=0.03). Conclusion: This study provides preliminary evidences that metabolic resistance potentially exerts a fitness cost on mating competiveness in resistant mosquitoes.

Functional Genomics Analysis of Horseweed (Conyza canadensis) with Special Reference to the Evolution of Non–Target-Site Glyphosate Resistance

Weed Science ◽  
2010 ◽  
Vol 58 (2) ◽  
pp. 109-117 ◽  
Author(s):  
Joshua S. Yuan ◽  
Laura L. G. Abercrombie ◽  
Yongwei Cao ◽  
Matthew D. Halfhill ◽  
Xin Zhou ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Molecular Mechanisms ◽  
Glyphosate Resistance ◽  
Resistance Mechanisms ◽  
Target Site ◽  
Environmentally Benign ◽  
Conyza Canadensis ◽  
Target Site Resistance ◽  
Weedy Species ◽  
Heterologous Microarray

The evolution of glyphosate resistance in weedy species places an environmentally benign herbicide in peril. The first report of a dicot plant with evolved glyphosate resistance was horseweed, which occurred in 2001. Since then, several species have evolved glyphosate resistance and genomic information about nontarget resistance mechanisms in any of them ranges from none to little. Here, we report a study combining iGentifier transcriptome analysis, cDNA sequencing, and a heterologous microarray analysis to explore potential molecular and transcriptomic mechanisms of nontarget glyphosate resistance of horseweed. The results indicate that similar molecular mechanisms might exist for nontarget herbicide resistance across multiple resistant plants from different locations, even though resistance among these resistant plants likely evolved independently and available evidence suggests resistance has evolved at least four separate times. In addition, both the microarray and sequence analyses identified non–target-site resistance candidate genes for follow-on functional genomics analysis.

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