Highly Efficient Site-Specific Mutagenesis in Malaria Mosquitoes Using CRISPR

Ming Li, Omar S. Akbari, and Bradley J. White (2017)

According to the latest WHO’s report, Malaria has infected 216 million people (almost 6 times the population of Canada) in 2016 and killed 445 000 people (a quarter of the Montrealers). It’s a major issue especially in sub-Saharan countries and US$ 3 billion (0.5% of the military budget of the USA) is invested every year to fight the disease.

Malaria is caused by Plasmodium parasites which are spread through infected female Anopheles mosquitoes.

Ming Li et al. conducted a study on those mosquitoes to develop a site-specific mutagenesis method using CRISPR/Cas9 system which enables to edit or modulate DNA sequences, allowing to study the function of genes in vivo. They designed a sgRNA (Small Guide RNA) to target one of the exon of the white gene. They chose this gene because the phenotype is easy to observe and the knockout of one allele with CRISPR/Cas9, despite it is recessive, would be sufficient on males. Indeed the gene is located on the X chromosome.

On this figure you can see the observation under a stereomicroscope for three different species of mosquitoes the wild-type red eyes and the unpigmented.

To obtain those results, they injected in mosquito eggs “cocktails” of sgRNA and Cas9 in different proportions to find the best compromise between the survival of the subjects and the change of phenotype as shown in this table.

Then, they crossed mosaic-eyed males they produced with females of an existing white-eye line to prove that the males where able to transmit their mutation. With 93% of the female progeny which had white-eyed phenotype, they proved that it works. They did the same thing crossing their mosaic males and females and obtained 83% of white-eyed. They also succeed in maintaining mutant lines for more than 15 generations, proving the mutation introduced with CRISPR/Cas9 is stable and that it could be a powerful tool to better understand Anopheles vector and better fight Malaria.