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The BBC article “Gene editing wipes out mosquitoes in the lab” discusses work using a gene editing technique to suppress populations of caged mosquitoes Anopheles gambiae, which transmits malaria. In 2016 were 216 million cases of malaria in 91 countries and 445000 people were killed globally. This study was published in journal Nature Biotechnology. A CRISPR-Cas9 gene drive was used to target doublesex gene to cutting down egg production, to eliminate the population. The study found that the gene doublesex (Agdsx) in the human malaria vector Anopheles gambiae contain 7 exons spanning an 85 regions of chromosome 2R, and is encoded by two transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), which regulate the differentiation of the two sexes.
The AgdsxF transcript contain a 5’ common segment with specific exon (exon 5-which is highly conserved, and it is only expressed in female mosquitoes), and 3’common region while the AgdsxM consists only the 5’ and 3’ common segments, and the specific region is transcribed as a noncoding 3′ untranslated region in females. The Pam (protospacer-adjacent motif) of the gRNA (highlighted in blue). Noncoding regions are white, coding regions of exons are shaded in black. To find out if is an acceptable target for a gene drive to eliminate the population of mosquitoes, the intron 4-exon 5 boundary of dsx was disrupted. The anopheles gambiae embryos were injected with a source of Cas9and a single-guide RNA (sgRNA) in combination with a template for homology-directed repair (HDR) to insert an eGFP transcription unit. To generate heterozygous and homozygous mutants scientists intercrossed transformed individuals. To determine the genotype diagnostic PCR was used with primers extended to the insertion site.
The study confirms that CRISPR-Cas9 sucessfully blocked the formation of functional AgdsxF. In the female homozygous mutants presents within the population there showed an intersex phenotype and complete sterility while male development was unaffected. Scientists found that the replacement of the 3xP3: GFP transcription unit with a dsxFCRISPRh allele will have a drastic effect of the reproductive output of population. The cage was set up with a wild-type mosquito population with heterozygous individuals carrying the dsxFCRISPRh. The study conclude that the drive reached maximum frequency by generation 7, by generation 8 no eggs were produced proving the population was sterile, and subsequently the population collapsed. Total egg output in each generation. Black arrow-population collapse completely.
The gene drive dsxFCRISPRh targeting exon 5 of dsx could lead a trial to successful population control. This drive has a lot of characteristic features like high inheritance bias, homozygous females are unproductive and did not bite, heterozygous individuals are fully productive which make this drive to succeed in future to field testing. With recommendations of the US National Academy of Sciences, further investigation needs to be strictly evaluated in natural landscapes with environmental conditions like the presence of predators, competitions for food.
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