RÉSUMÉ
Culex mosquitoes pose a significant public health threat as vectors for a variety of diseases including West Nile virus and lymphatic filariasis, and transmit pathogens threatening livestock, companion animals, and endangered birds. Rampant insecticide resistance makes controlling these mosquitoes challenging and necessitates the development of new control strategies. Gene drive technologies have made significant progress in other mosquito species, although similar advances have been lagging in Culex. Here we test a CRISPR-based homing gene drive for Culex quinquefasciatus, and show that the inheritance of two split-gene-drive transgenes, targeting different loci, are biased in the presence of a Cas9-expressing transgene although with modest efficiencies. Our findings extend the list of disease vectors where engineered homing gene drives have been demonstrated to include Culex alongside Anopheles and Aedes, and pave the way for future development of these technologies to control Culex mosquitoes.
Sujet(s)
Aedes , Culex , Forçage génétique , Animaux , Culex/génétique , Vecteurs moustiques/génétique , Aedes/génétique , Vecteurs de maladiesRÉSUMÉ
Culex mosquitoes pose a significant public health threat as vectors for a variety of diseases including West Nile virus and lymphatic filariasis, and transmit pathogens threatening livestock, companion animals, and endangered birds. Rampant insecticide resistance makes controlling these mosquitoes challenging and necessitates the development of new control strategies. Gene drive technologies have made significant progress in other mosquito species, although similar advances have been lagging in Culex. Here we test the first CRISPR-based homing gene drive for Culex quinquefasciatus, demonstrating the possibility of using this technology to control Culex mosquitoes. Our results show that the inheritance of two split-gene-drive transgenes, targeting different loci, are biased in the presence of a Cas9-expressing transgene although with modest efficiencies. Our findings extend the list of disease vectors where engineered homing gene drives have been demonstrated to include Culex alongside Anopheles and Aedes, and pave the way for future development of these technologies to control Culex mosquitoes.
RÉSUMÉ
OBJECTIVES: To assess natural history and 12-month change of a series of scales and functional outcome measures in a cohort of 117 patients with primary mitochondrial myopathy (PMM). METHODS: Twelve months follow-up data of 117 patients with PMM were collected. We analysed the 6-min walk test (6MWT), timed up-and-go test (× 3) (3TUG), five-times sit-to-stand test (5XSST), timed water swallow test (TWST), and test of masticating and swallowing solids (TOMASS) as functional outcome measures; the Fatigue Severity Scale and West Haven-Yale Multidimensional pain inventory as patient-reported outcome measures. PMM patients were divided into three phenotypic categories: mitochondrial myopathy (MiMy) without extraocular muscles involvement, pure chronic progressive external ophthalmoplegia (PEO) and PEO&MiMy. As 6MWT is recognized to have significant test-retest variability, we calculated MCID (minimal clinically important difference) as one third of baseline 6 min walking distance (6MWD) standard deviation. RESULTS: At 12-month follow-up, 3TUG, 5XSST and FSS were stable, while TWST and the perceived pain severity (WHYMPI) worsened. 6MWD significantly increased in the entire cohort, especially in the higher percentiles and in PEO patients, while was substantially stable in the lower percentile (< 408 m) and MiMy patients. This increase in 6MWD was considered not significant, as inferior to MCID (33.3 m). NMDAS total score showed a slight but significant decline at 12 months (0.9 point). The perceived pain severity significantly worsened. Patients with PEO performed better in functional measures than patients with PEO&MiMy or MiMy, and had lower values of NMDAS. CONCLUSIONS: PMM patients showed a slow global decline valued by NMDAS at 12 months; 6MWT was a more reliable measurement below 408 m, substantially stable at 12 months. PEO patients had better motor performance and lower NMDAS than PEO&MiMy and MiMy also at 12 months of follow-up.
Sujet(s)
Myopathies mitochondriales , Ophtalmoplégie externe progressive , Humains , Études de suivi , Test de marche/méthodes , Myopathies mitochondriales/complications , Myopathies mitochondriales/diagnostic , Facteurs temps , Marche à piedRÉSUMÉ
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based "active genetic" elements developed in 2015 bypassed the fundamental rules of traditional genetics. Inherited in a super-Mendelian fashion, such selfish genetic entities offered a variety of potential applications including: gene-drives to disseminate gene cassettes carrying desired traits throughout insect populations to control disease vectors or pest species, allelic drives biasing inheritance of preferred allelic variants, neutralizing genetic elements to delete and replace or to halt the spread of gene-drives, split-drives with the core constituent Cas9 endonuclease and guide RNA (gRNA) components inserted at separate genomic locations to accelerate assembly of complex arrays of genetic traits or to gain genetic entry into novel organisms (vertebrates, plants, bacteria), and interhomolog based copying systems in somatic cells to develop tools for treating inherited or infectious diseases. Here, we summarize the substantial advances that have been made on all of these fronts and look forward to the next phase of this rapidly expanding and impactful field.
Sujet(s)
Systèmes CRISPR-Cas , Forçage génétique , Animaux , Systèmes CRISPR-Cas/génétique , Édition de gène , Modes de transmission héréditaire , /génétiqueRÉSUMÉ
CRISPR-based gene-drives have been proposed for managing insect populations, including disease-transmitting mosquitoes, due to their ability to bias their inheritance toward super-Mendelian rates (>50%). Current technologies use a Cas9 that introduces DNA double-strand breaks into the opposing wild-type allele to replace it with a copy of the gene-drive allele via DNA homology-directed repair. However, the use of different Cas9 versions is unexplored, and alternative approaches could increase the available toolkit for gene-drive designs. Here, we report a gene-drive that relies on Cas9 nickases that generate staggered paired nicks in DNA to propagate the engineered gene-drive cassette. We show that generating 5' overhangs in the system yields efficient allelic conversion. The nickase gene-drive arrangement produces large, stereotyped deletions that are advantageous to eliminate viable animals carrying small mutations when targeting essential genes. Our nickase approach should expand the repertoire for gene-drive arrangements aimed at applications in mosquitoes and beyond.
Sujet(s)
Deoxyribonuclease I , Forçage génétique , Animaux , Systèmes CRISPR-Cas/génétique , ADN , Deoxyribonuclease I/métabolisme , Drosophila/métabolisme , Édition de gèneRÉSUMÉ
Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. However, end-joining repair pathways often outcompete HDR and introduce insertions and deletions of bases (indels) at the DSB site, decreasing precision outcomes. It has been shown that indel sequences for a given DSB site are reproducible and can even be predicted. Here, we report a general strategy (the "double tap" method) to improve HDR-mediated precision genome editing efficiencies that takes advantage of the reproducible nature of indel sequences. The method simply involves the use of multiple gRNAs: a primary gRNA that targets the wild-type genomic sequence, and one or more secondary gRNAs that target the most common indel sequence(s), which in effect provides a "second chance" at HDR-mediated editing. This proof-of-principle study presents the double tap method as a simple yet effective option for enhancing precision editing in mammalian cells.
Sujet(s)
Édition de gène , , Animaux , Systèmes CRISPR-Cas/génétique , Réparation de l'ADN par jonction d'extrémités , Édition de gène/méthodes , Mammifères/génétique , /génétique , /métabolisme , Réparation de l'ADN par recombinaisonRÉSUMÉ
Homing CRISPR gene drives could aid in curbing the spread of vector-borne diseases and controlling crop pest and invasive species populations due to an inheritance rate that surpasses Mendelian laws. However, this technology suffers from resistance alleles formed when the drive-induced DNA break is repaired by error-prone pathways, which creates mutations that disrupt the gRNA recognition sequence and prevent further gene-drive propagation. Here, we attempt to counteract this by encoding additional gRNAs that target the most commonly generated resistance alleles into the gene drive, allowing a second opportunity at gene-drive conversion. Our presented "double-tap" strategy improved drive efficiency by recycling resistance alleles. The double-tap drive also efficiently spreads in caged populations, outperforming the control drive. Overall, this double-tap strategy can be readily implemented in any CRISPR-based gene drive to improve performance, and similar approaches could benefit other systems suffering from low HDR frequencies, such as mammalian cells or mouse germline transformations.
Sujet(s)
Forçage génétique , Allèles , Animaux , Systèmes CRISPR-Cas/génétique , Cellules germinales , Mammifères/génétique , Souris , /génétiqueRÉSUMÉ
INTRODUCTION: Both prevalence and clinical features of the various movement disorders in adults with primary mitochondrial diseases are unknown. METHODS: Based on the database of the "Nation-wide Italian Collaborative Network of Mitochondrial Diseases", we reviewed the clinical, genetic, neuroimaging and neurophysiological data of adult patients with primary mitochondrial diseases (n = 764) where ataxia, myoclonus or other movement disorders were part of the clinical phenotype. RESULTS: Ataxia, myoclonus and movement disorders were present in 105/764 adults (13.7%), with the onset coinciding or preceding the diagnosis of the mitochondrial disease in 49/105 (46.7%). Ataxia and parkinsonism were the most represented, with an overall prevalence at last follow-up of 59.1% and 30.5%, respectively. Hyperkinetic movement disorders were reported in 15.3% at last follow-up, being the less common reported movement disorders. The pathogenic m.8344A > G and POLG variants were always associated with a movement disorder, while LHON variants and mtDNA single deletions were more commonly found in the subjects who did not present a movement disorder. The most common neuroimaging features were cortical and/or cerebellar atrophy, white matter hyperintensities, basal ganglia abnormalities and nigro-striatal degeneration. Almost 70% of patients with parkinsonism responded to dopaminergic therapy, mainly levodopa, and 50% with myoclonus were successfully treated with levetiracetam. CONCLUSION: Movement disorders, mainly ataxia and parkinsonism, are important findings in adult primary mitochondrial diseases. This study underlies the importance of looking for a mitochondrial etiology in the diagnostic flowchart of a movement disorder and may help direct genetic screening in daily practice.
Sujet(s)
Maladies mitochondriales , Troubles de la motricité , Myoclonie , Syndromes parkinsoniens , Humains , Maladies mitochondriales/complications , Maladies mitochondriales/épidémiologie , Maladies mitochondriales/génétique , Troubles de la motricité/diagnostic , Troubles de la motricité/épidémiologie , Troubles de la motricité/génétique , Syndromes parkinsoniens/complications , Syndromes parkinsoniens/épidémiologie , Syndromes parkinsoniens/génétique , PhénotypeRÉSUMÉ
Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a "gene drive," to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) mechanisms to form an "insertion/deletion" (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology-directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate gene conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of "super-mendelian" inheritance from both male and female mice.
Sujet(s)
Protéine-9 associée à CRISPR/métabolisme , Conversion des gènes/génétique , Édition de gène/méthodes , Animaux , Protéine-9 associée à CRISPR/génétique , Systèmes CRISPR-Cas/génétique , Cassures double-brin de l'ADN , Réparation de l'ADN/génétique , Femelle , Expression des gènes/génétique , Régulation de l'expression des gènes au cours du développement/génétique , Génie génétique/méthodes , Cellules germinales/métabolisme , Mâle , Méiose/génétique , Souris , /génétique , Réparation de l'ADN par recombinaison/génétiqueRÉSUMÉ
Culex quinquefasciatus mosquitoes are a globally widespread vector of several human and animal pathogens. Their biology and behavior allow them to thrive in proximity to urban areas, rendering them a constant public health threat. Their mixed bird/mammal feeding behavior further offers a vehicle for zoonotic pathogens transmission to people and, separately, poses a threat to the conservation of insular birds. The advent of CRISPR has led to the development of novel technologies for the genetic engineering of wild mosquito populations. Yet, research into Cx. quinquefasciatus has been lagging compared to other disease vectors. Here, we use this tool to disrupt a set of five pigmentation genes in Cx. quinquefasciatus that, when altered, lead to visible, homozygous-viable phenotypes. We further validate this approach in separate laboratories and in two distinct strains of Cx. quinquefasciatus that are relevant to potential future public health and bird conservation applications. We generate a double-mutant line, demonstrating the possibility of sequentially combining multiple such mutations in a single individual. Lastly, we target two loci, doublesex in the sex-determination pathway and proboscipedia, a hox gene, demonstrating the flexibility of these methods applied to novel targets. Our work provides a platform of seven validated loci that could be used for targeted mutagenesis in Cx. quinquefasciatus and the future development of genetic suppression strategies for this species. Furthermore, the mutant lines generated here could have widespread utility to the research community using this model organism, as they could be used as targets for transgene delivery, where a copy of the disrupted gene could be included as an easily scored transgenesis marker.
Sujet(s)
Systèmes CRISPR-Cas , Clustered regularly interspaced short palindromic repeats , Culex/génétique , Édition de gène , Techniques de knock-out de gènes , Génie génétique , Animaux , Culicidae/génétique , Édition de gène/méthodes , Ciblage de gène , Génie génétique/méthodes , Locus génétiques , Microinjections , Mutation , Phénotype ,RÉSUMÉ
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.
Sujet(s)
Systèmes CRISPR-Cas/génétique , Réparation de l'ADN par jonction d'extrémités , Conversion des gènes , Édition de gène/méthodes , Réparation de l'ADN par recombinaison , Animaux , Animal génétiquement modifié , Drosophila/génétique , Études de faisabilité , Femelle , Locus génétiques , Thérapie génétique/méthodes , Cellules HEK293 , Humains , Mâle , Modèles animaux , Protéines proto-oncogènes c-myc/génétiqueRÉSUMÉ
Culex mosquitoes are a global vector for multiple human and animal diseases, including West Nile virus, lymphatic filariasis, and avian malaria, posing a constant threat to public health, livestock, companion animals, and endangered birds. While rising insecticide resistance has threatened the control of Culex mosquitoes, advances in CRISPR genome-editing tools have fostered the development of alternative genetic strategies such as gene drive systems to fight disease vectors. However, though gene-drive technology has quickly progressed in other mosquitoes, advances have been lacking in Culex. Here, we develop a Culex-specific Cas9/gRNA expression toolkit and use site-directed homology-based transgenesis to generate and validate a Culex quinquefasciatus Cas9-expressing line. We show that gRNA scaffold variants improve transgenesis efficiency in both Culex quinquefasciatus and Drosophila melanogaster and boost gene-drive performance in the fruit fly. These findings support future technology development to control Culex mosquitoes and provide valuable insight for improving these tools in other species.
Sujet(s)
Systèmes CRISPR-Cas/génétique , Culex/génétique , Forçage génétique/méthodes , Lutte contre les moustiques/méthodes , Vecteurs moustiques/génétique , Animaux , Animal génétiquement modifié , Drosophila melanogaster/génétique , Femelle , Résistance aux insecticides , Mâle , Mutagenèse dirigée/méthodes , /génétiqueRÉSUMÉ
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.
Sujet(s)
Anopheles/génétique , Paludisme/parasitologie , Allèles , Animaux , Protéine-9 associée à CRISPR/métabolisme , Femelle , Génétique des populations , Protéines à fluorescence verte/métabolisme , Hétérozygote , Modes de transmission héréditaire/génétique , Kynurenine 3-monooxygenase/génétique , Mâle , Modèles génétiques , Mosaïcisme , Phénotype , Phylogenèse , /métabolismeRÉSUMÉ
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.
Sujet(s)
Délétion de gène , Forçage génétique , Animaux , Protéine-9 associée à CRISPR/métabolisme , Chromosomes/génétique , Drosophila melanogaster/génétique , Femelle , Protéines à fluorescence verte/métabolisme , Modes de transmission héréditaire/génétique , Mutagenèse/génétique , /génétique , TransgènesRÉSUMÉ
Synthetic CRISPR-based gene-drive systems have tremendous potential in public health and agriculture, such as for fighting vector-borne diseases or suppressing crop pest populations. These elements can rapidly spread in a population by breaching the inheritance limit of 50% dictated by Mendel's law of gene segregation, making them a promising tool for population engineering. However, current technologies lack control over their propagation capacity, and there are important concerns about potential unchecked spreading. Here, we describe a gene-drive system in Drosophila that generates an analog inheritance output that can be tightly and conditionally controlled to between 50% and 100%. This technology uses a modified SpCas9 that responds to a synthetic, orally available small molecule, fine-tuning the inheritance probability. This system opens a new avenue to feasibility studies for spatial and temporal control of gene drives using small molecules.
Sujet(s)
Drosophila melanogaster/génétique , Forçage génétique , Modes de transmission héréditaire/génétique , Bibliothèques de petites molécules/métabolisme , Animaux , Animal génétiquement modifié , Protéine-9 associée à CRISPR/métabolisme , Préparations pharmaceutiquesRÉSUMÉ
Mitochondrial medicine is a field that expanded exponentially in the last 30 years. Individually rare, mitochondrial diseases as a whole are probably the most frequent genetic disorder in adults. The complexity of their genotype-phenotype correlation, in terms of penetrance and clinical expressivity, natural history and diagnostic algorithm derives from the dual genetic determination. In fact, in addition to the about 1.500 genes encoding mitochondrial proteins that reside in the nuclear genome (nDNA), we have the 13 proteins encoded by the mitochondrial genome (mtDNA), for which 22 specific tRNAs and 2 rRNAs are also needed. Thus, besides Mendelian genetics, we need to consider all peculiarities of how mtDNA is inherited, maintained and expressed to fully understand the pathogenic mechanisms of these disorders. Yet, from the initial restriction to the narrow field of oxidative phosphorylation dysfunction, the landscape of mitochondrial functions impinging on cellular homeostasis, driving life and death, is impressively enlarged. Finally, from the clinical standpoint, starting from the neuromuscular field, where brain and skeletal muscle were the primary targets of mitochondrial dysfunction as energy-dependent tissues, after three decades virtually any subspecialty of medicine is now involved. We will summarize the key clinical pictures and pathogenic mechanisms of mitochondrial diseases in adults.
Sujet(s)
Maladies mitochondriales/génétique , Adulte , Réparation de l'ADN/génétique , ADN mitochondrial/génétique , Génome mitochondrial/génétique , Humains , Syndrome MELAS/diagnostic , Syndrome MELAS/génétique , Mitochondries/génétique , Mitochondries/métabolisme , Maladies mitochondriales/diagnostic , Mutation/génétiqueRÉSUMÉ
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RÉSUMÉ
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.
Sujet(s)
Forçage génétique/méthodes , Génétique des populations/méthodes , Allèles , Animaux , Animal génétiquement modifié , Séquence nucléotidique , Systèmes CRISPR-Cas , Diptera , Écosystème , Femelle , Édition de gène , Gènes liés au chromosome X , Mâle , Modèles théoriques , /génétique , TransgènesRÉSUMÉ
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 Drosophila melanogaster 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 Drosophila 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.