Ultra-conserved sequences in the genomes of highly diverse <i>Anopheles</i> mosquitoes, with implications for malaria vector control

OʼLoughlin, Samantha; Forster, Annie J; Fuchs, Silke; Dottorini, Tania; Nolan, Tony; Crisanti, Andrea; Burt, Austin

doi:10.1093/g3journal/jkab086

Cited by 3 publications

(4 citation statements)

References 64 publications

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“…A recent analysis of ultra-conserved elements (UCEs) from 21 different Anopheles species and search of functional annotations of genes containing UCEs by O'Loughlin et al [12] identified ultra-conserved targets potentially affecting female fertility or lethal recessive phenotype that could be suitable targets for vector control. Amongst the list of potential targets, the AGAP029113 gene is of interest as it contains multiple ultra-conserved sites with a total of 140 invariant sites.…”

Section: Agap029113 Is a Haplosufficient Essential Gene In Anopheles Gambiaementioning

confidence: 99%

“…Our ability to identify novel genes containing gene drive targets that show high functional constraint has been greatly enhanced by genome resequencing efforts that have covered species across the Anopheles genus, spanning over 100 million years on an evolutionary timescale, as well as wild caught individuals of the closely related Anopheles gambiae and Anopheles coluzzii species [9][10][11]. A recent analysis of genomic data from 21 Anopheles species revealed over 8000 ultra-conserved sites (100% identity) of sufficient length (18bp; corresponding to the minimal sequence recognised by a guide RNA) to be targetable by homing-based gene drives [12]. It is to be expected that among this set of sequences there will be variation in their tolerance for changes to their underlying biological functions.…”

Section: Introductionmentioning

confidence: 99%

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Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation

et al. 2021

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CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying (‘homing’) therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel adult lethal gene drive (ALGD) in the malaria vector Anopheles gambiae, targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations.

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Section: Agap029113 Is a Haplosufficient Essential Gene In Anopheles Gambiaementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation

et al. 2021

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“…Researchers are examining several different and potentially complementary approaches to delay the appearance of resistance, including: aiming to disrupt multiple female fertility genes and/or multiple sequences within the same gene, utilizing promoters that bias toward homologous DNA repair and thus reduce the opportunity for mutation; and, aiming to disrupt genes that are highly conserved in the target mosquito species and/or cannot tolerate changes in their sequence without causing severe harm to the mosquito that would make a mutation less likely to be inherited (for review, see [72]). Methods are being developed to evaluate candidate gene drive targets for their resilience to resistance (e.g., [83][84][85]). For example, cage trials of a gene drive targeting doublesex described above found complete population suppression in A. gambiae mosquitoes with no evidence of resistance, presumably due to functional constraints in the target sequence.…”

Section: Challenges For Implementation Of Genetic Biocontrolmentioning

confidence: 99%

The Promise and Challenge of Genetic Biocontrol Approaches for Malaria Elimination

James

Santos

2023

TropicalMed

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Malaria remains an ongoing public health challenge, with over 600,000 deaths in 2021, of which approximately 96% occurred in Africa. Despite concerted efforts, the goal of global malaria elimination has stalled in recent years. This has resulted in widespread calls for new control methods. Genetic biocontrol approaches, including those focused on gene-drive-modified mosquitoes (GDMMs), aim to prevent malaria transmission by either reducing the population size of malaria-transmitting mosquitoes or making the mosquitoes less competent to transmit the malaria parasite. The development of both strategies has advanced considerably in recent years, with successful field trials of several biocontrol methods employing live mosquito products and demonstration of the efficacy of GDMMs in insectary-based studies. Live mosquito biocontrol products aim to achieve area-wide control with characteristics that differ substantially from current insecticide-based vector control methods, resulting in some different considerations for approval and implementation. The successful field application of current biocontrol technologies against other pests provides evidence for the promise of these approaches and insights into the development pathway for new malaria control agents. The status of technical development as well as current thinking on the implementation requirements for genetic biocontrol approaches are reviewed, and remaining challenges for public health application in malaria prevention are discussed.

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“…The increasing genomic resources available have allowed conservation analysis to be performed across the Anopheles species complex to identify regions that are highly conserved ( Kranjc et al, 2021 ; O’Loughlin et al, 2021 ). The use of these bioinformatic resources can help guide where to target a gene drive, such that it is at a genomic site that could prove intolerant of mutations.…”

Section: Lessons Learnt Towards the Next Generation Of Gene Drivesmentioning

confidence: 99%

Driving down malaria transmission with engineered gene drives

et al. 2022

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The last century has witnessed the introduction, establishment and expansion of mosquito-borne diseases into diverse new geographic ranges. Malaria is transmitted by female Anopheles mosquitoes. Despite making great strides over the past few decades in reducing the burden of malaria, transmission is now on the rise again, in part owing to the emergence of mosquito resistance to insecticides, antimalarial drug resistance and, more recently, the challenges of the COVID-19 pandemic, which resulted in the reduced implementation efficiency of various control programs. The utility of genetically engineered gene drive mosquitoes as tools to decrease the burden of malaria by controlling the disease-transmitting mosquitoes is being evaluated. To date, there has been remarkable progress in the development of CRISPR/Cas9-based homing endonuclease designs in malaria mosquitoes due to successful proof-of-principle and multigenerational experiments. In this review, we examine the lessons learnt from the development of current CRISPR/Cas9-based homing endonuclease gene drives, providing a framework for the development of gene drive systems for the targeted control of wild malaria-transmitting mosquito populations that overcome challenges such as with evolving drive-resistance. We also discuss the additional substantial works required to progress the development of gene drive systems from scientific discovery to further study and subsequent field application in endemic settings.

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Ultra-conserved sequences in the genomes of highly diverse Anopheles mosquitoes, with implications for malaria vector control

Cited by 3 publications

References 64 publications

Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation

Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation

The Promise and Challenge of Genetic Biocontrol Approaches for Malaria Elimination

Driving down malaria transmission with engineered gene drives

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