Hammond et al.’s just published paper in Nature Biotechnology is the second paper in a month demonstrating effective CRISPR-based gene drive in a mosquito vector of malaria. In this case the main malaria vector in Africa, Anopheles gambiae.
Gene drive as a control for vectors of human disease can be used in two ways: prevention of disease transmission, or suppression of the vector population. Hammond et al. chose to pursue the latter method, and identified three candidate genes to test for haplosufficient roles in female fertility. Rather than risk employing gene drive to prove the function of their candidates, the researchers mutated each gene with a GFP-marked attP insertion. As expected, each of the three loci only caused infertility in homozygous mutant females; males and heterozygous females showed normal fertility, which is necessary for the drive system in these loci to spread. So, the authors proceeded with creating and testing lines with CRISPR-based gene drive.
The attP sites allowed Hammond and colleagues to insert gene-drive cassettes, which included an RFP marker, into each of the mutation sites, maintaining the original infertility phenotypes. Conversion rates of wildtype alleles to RFP+ varied by locus and across generations, but remained consistently high, between 69-98%. Caged population tests of the drive at the most efficient locus showed increases in allele frequency over four generations, in spite of the fitness cost carried by the impacts on female fertility.
However, the authors note three important caveats. First, they show that occasionally, non-conversion mutations are capable of arising, blocking future CRISPR activity at those sites. They even discovered two mutants with a 6 bp deletion, which would potentially leave the gene intact, preventing loss of the allele due to infertility. Such mutations could potentially flourish in the population in the presence of the drive, countering its efficacy. Second, they note significant reduction in fertility of females heterozygous for the drive at each of the three loci, suggesting a negative impact from the expression of Cas9. The authors suggest this might be overcome with a more precise promoter. Finally, they use modeling to show that the fitness costs at two loci outweighed the power of the drive, preventing the drive alleles from surviving in wild populations indefinitely.
While this is the second paper to demonstrate CRISPR gene drive in mosquitoes, it is the first to use drive for population suppression. In doing so, it demonstrates the importance of coupling gene-drive for any purpose with insertions into fitness genes in order to sustain the efficacy of the drive for longer. Even then, this work suggests that mutations are able to prevent even powerful drives from altering a population permanently. Future work should consider both the persistence of the drive through many generations, and the possible benefits of even short-term drives on human health.
Hammond, A., Galizi, R., Kyrou, K., Simoni, A., Siniscalchi, C. et al., 2015 A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae. Nat Biotech advance online publication: doi: 10.1038/nbt.3439
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