Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of <i>Anopheles</i> mosquitoes

Vitale, Matteo; Leo, Chiara; Courty, Thomas; Kranjc, Nace; Connolly, John B.; Morselli, Giulia; Bamikole, Christopher; Haghighat-Khah, Roya Elaine; Bernardini, Federica; Fuchs, Silke

doi:10.1080/20477724.2022.2100192

“…This genomic block includes an approximately 35‐Mb pericentromeric region, characterized by extremely low recombination rates (White et al, 2010 ). Coupled with the transgene location in the band 2R‐19D, near the centromere of chromosome 2 (Vitale et al, 2022 ) and the high linkage disequilibrium of the neighbouring pericentromeric environment, the artificial selection of the adult females carrying the transgene as maternal donors at each stage of the serial backcrossing strategy may have contributed to retain G3 haplotypes in such a genomic region. The incomplete introgression of the kdr_r resistant allele associated with the BF‐specific 2L‐island haplotype in the final transgenic BF_BC6 offspring is consistent with this prediction.…”

Section: Discussionmentioning

confidence: 99%

“…When Ag(PMB)1 males are crossed to wild‐type females, the expression of I‐PpoI endonuclease causes the meiotic cleavage of X‐linked rDNA repeats, affecting the X‐bearing sperm to fertilize the female oocyte, leading to fertile offspring with a 95% male bias and a 50% transgene inheritance. Although a single copy integration of the [3xP3‐DsRed]β2‐eGFP::I‐PpoI‐124L transgene has been initially attested in the chromosome band 3R‐36D for Ag(PMB)1 strain (Galizi et al, 2014 ), this integration site has been recently revised and re‐assigned to a poorly annotated centromeric region of chromosome 2R 19D (Vitale et al, 2022 ). Transgenic lines are typically developed in laboratory strains, and in the case of Ag(PMB)1 developed in the G3 wildtype colony of Anopheles mosquitoes, which was isolated in The Gambia (Africa) and has been maintained in the laboratory for approximately 47 years (Malaria Research and Reference Reagent Resource Center, Atlanta, USA; stock number MRA‐112).…”

Section: Introductionmentioning

confidence: 99%

Introgression of a synthetic sex ratio distortion transgene into different genetic backgrounds ofAnopheles coluzzii

Pollegioni

¹

,

Persampieri

²

,

Minuz

³

et al. 2022

Insect Molecular Biology

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The development of genetically modified mosquitoes (GMM) and their subsequent field release offers innovative approaches for vector control of malaria. A non‐gene drive self‐limiting male‐bias Ag(PMB)1 strain has been developed in a 47‐year‐old laboratory G3 strain of Anopheles gambiae s.l. When Ag(PMB)1 males are crossed to wild‐type females, expression of the endonuclease I‐PpoI during spermatogenesis causes the meiotic cleavage of the X chromosome in sperm cells, leading to fertile offspring with a 95% male bias. However, World Health Organization states that the functionality of the transgene could differ when inserted in different genetic backgrounds of Anopheles coluzzii which is currently a predominant species in several West‐African countries and thus a likely recipient for a potential release of self‐limiting GMMs. In this study, we introgressed the transgene from the donor Ag(PMB)1 by six serial backcrosses into two recipient colonies of An. coluzzii that had been isolated in Mali and Burkina Faso. Scans of informative Single Nucleotide Polymorphism (SNP) markers and whole‐genome sequencing analysis revealed a nearly complete introgression of chromosomes 3 and X, but a remarkable genomic divergence in a large region of chromosome 2 between the later backcrossed (BC6) transgenic offspring and the recipient paternal strains. These findings suggested to extend the backcrossing breeding strategy beyond BC6 generation and increasing the introgression efficiency of critical regions that have ecological and epidemiological implications through the targeted selection of specific markers. Disregarding differential introgression efficiency, we concluded that the phenotype of the sex ratio distorter is stable in the BC6 introgressed An. coluzzii strains.

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“…The expression of I-Ppol endonuclease in male germline leads to approximately 95% male offspring [ 32 ]. The transgene is located on Chromosome 2R band 19D [ 82 ]. The strain named Vasa2:Cas9 harbours a genetic construct where Vasa2 5’ regulatory sequences mediate the expression of Cas9 endonuclease in the germline of both male and female mosquitoes [ 52 , 83 ].…”

Section: Methodsmentioning

confidence: 99%

“…al. 2022 [ 82 ]. Only Nanopore reads between 1kbs and 100kbs were selected as input for RedKmer pipeline.…”

Section: Methodsmentioning

confidence: 99%

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Y chromosome shredding in Anopheles gambiae: Insight into the cellular dynamics of a novel synthetic sex ratio distorter

Vitale,

Kranjc,

Leigh

et al. 2024

PLoS Genet

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Despite efforts to explore the genome of the malaria vector Anopheles gambiae, the Y chromosome of this species remains enigmatic. The large number of repetitive and heterochromatic DNA sequences makes the Y chromosome exceptionally difficult to fully assemble, hampering the progress of gene editing techniques and functional studies for this chromosome. In this study, we made use of a bioinformatic platform to identify Y-specific repetitive DNA sequences that served as a target site for a CRISPR/Cas9 system. The activity of Cas9 in the reproductive organs of males caused damage to Y-bearing sperm without affecting their fertility, leading to a strong female bias in the progeny. Cytological investigation allowed us to identify meiotic defects and investigate sperm selection in this new synthetic sex ratio distorter system. In addition, alternative promoters enable us to target the Y chromosome in specific tissues and developmental stages of male mosquitoes, enabling studies that shed light on the role of this chromosome in male gametogenesis. This work paves the way for further insight into the poorly characterised Y chromosome of Anopheles gambiae. Moreover, the sex distorter strain we have generated promises to be a valuable tool for the advancement of studies in the field of developmental biology, with the potential to support the progress of genetic strategies aimed at controlling malaria mosquitoes and other pest species.

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Y chromosome shredding inAnopheles gambiae: insight into the cellular dynamics of a novel synthetic sex ratio distorter

Vitale,

Kranjc,

Leigh

et al. 2024

Preprint

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0

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Despite efforts to explore the genome of the malaria vector Anopheles gambiae, the Y chromosome of this species remains enigmatic. The large number of repetitive and heterochromatic DNA sequences makes the Y chromosome exceptionally difficult to fully assemble, hampering the progress of gene editing techniques and functional studies for this chromosome. In this study, we made use of a bioinformatic platform to identify Y-specific repetitive DNA sequences that served as a target site for a CRISPR/Cas9 system. The activity of Cas9 in the reproductive organs of males caused damage to Y-bearing sperm without affecting their fertility, leading to a strong female bias in the progeny. Cytological investigation allowed us to identify meiotic defects and investigate sperm selection in this new synthetic sex ratio distorter system. In addition, alternative promoters enable us to target the Y chromosome in specific tissues and developmental stages of male mosquitoes, enabling studies that shed light on the role of this chromosome in male gametogenesis. This work paves the way for further insight into the poorly characterised Y chromosome of Anopheles gambiae. Moreover, the sex distorter strain we have generated promises to be a valuable tool for the advancement of studies in the field of developmental biology, with the potential to support the progress of genetic strategies aimed at controlling malaria mosquitoes and other pest species.

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Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes

Cited by 4 publications

References 31 publications

Introgression of a synthetic sex ratio distortion transgene into different genetic backgrounds ofAnopheles coluzzii

Introgression of a synthetic sex ratio distortion transgene into different genetic backgrounds ofAnopheles coluzzii

Y chromosome shredding in Anopheles gambiae: Insight into the cellular dynamics of a novel synthetic sex ratio distorter

Y chromosome shredding inAnopheles gambiae: insight into the cellular dynamics of a novel synthetic sex ratio distorter

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