Gene drives meet germline biology
Promoters gonna promote
I’ve written previously about gene drive technology, which allows gene editing of entire species. This works by engineering alleles that are inherited at greater than the expected Mendelian frequency of 50%. One example is a CRISPR gene introduced into mosquitoes that copies itself to all offspring, while making only the female offspring sterile. This results in an increase in the proportion of males, shortly followed by a population crash.
I wrote “Gene drives: why the wait?” nearly four years ago, and since then we’re still waiting for gene drive deployment. Mainly this is due to political roadblocks, but there is also the concern that the gene drives will lose efficacy and generate resistance in the target species.
Mechanisms of resistance
Resistance to gene drives is an undesired side effect of their mechanism of spreading. Sometimes instead of copying the drive allele over into the wild-type allele, the drive breaks the wild-type allele in such a way that it generates a resistant allele. This is usually due to a repair pathway called non-homologous end joining (NHEJ), which competes with the deired homology-directed repair (HDR).
Importantly, HDR and NHEJ happen at different rates in different cell types. During meiosis I in the germline, HDR is extremely efficient, because that’s how cells perform meiotic recombination. However, earlier or later in germ cell development, there is less HDR and more NHEJ. So, restricting drive expression to meiotic germ cells is key to reducing resistance.
Furthermore, gene drive expression in somatic cells is also undesirable. Although this does not cause resistance (since DNA from somatic cells is not passed down to offspring), leaky somatic expression can reduce the health of the mosquito, making it very unsexy to its fellow bugs. A gene drive whose carriers don’t mate won’t spread.
Choosing the right promoter
So, how can a gene drive be regulated to only express itself during meiosis?
The key is using a promoter that’s only expressed in meiotic cells. Promoters are pieces of DNA that contain regulatory sequences directing transcription of the downstream gene. These sequences are often the binding sites for cell-type-specific regulatory factors.
Gene drive researchers have identified several naturally occurring promoters from genes that are expressed in germline cells (such as nanos), and these have been standard in the field for several years. However, there is still room for improvement, which brings me to the current study.
Researchers from Peking University (Wu et al.) studied the performance of 35 gene drive designs with different promoters, using Drosophila as a model organism for insects. They analyzed the drive performance and resistance generation of each design, and correlated it with expression of the promoter in different tissues of the fly. Drosophila is the best-understood insect in biology, and there is a lot of information about gene expression across cell types and developmental stages.
Three key takeaways of the study:
Integrating the drive into a germline-specific gene (rather than integration at an intergenic site with a germline-specific drive promoter) gives better specificity for germline expression. This is because the overall chromatin context matters, not just the promoter.1
Promoters that were more highly expressed in germline cells had better drive efficiencies. However, expression that is too high in female germline cells causes resistance allele formation, so a narrow range of expression is best for drives that act in females.2 The researchers identified several promoters that worked well in Drosophila, including several that were expressed in male germ cells just prior to meiosis.
Besides the promoter, the 3’ untranslated region of the drive allele is also important for driving germline-specific expression. The authors didn’t investigate this very much, but it could be a fruitful avenue of research.
Future developments
Based on my own experience designing meiosis-specific gene expression reporters, I think that the gene drive field definitely has room for large improvements in specificity. The main bottleneck is likely training data: it’s hard to perform single cell sequencing on insects (because of cell walls making it hard to isolate single cells),3 and germ cells tend to be rare populations.4 Drosophila is really the only organism with high quality datasets. I think it would be well worth studying germline gene expression in mosquitoes and screwworms.
Furthermore, I previously wrote about the possibility of designing synthetic regulatory sequences that achieve greater cell type specificity than natural promoters:
I think this approach could be quite useful for gene drives, and the field of synthetic regulatory elements has advanced a lot since I wrote that post.
Overall, better gene drive designs will help in the fight against mosquitoes, screwworms, and other nasty bugs. Hopefully the political will exists to deploy them.
However, most drives are limited in where they can be integrated into the genome, so this finding may be hard to apply.
For a drive that causes female sterility this would not be an issue.
Isolating cell nuclei and performing single nucleus RNA-seq is often used as an alternative, and this was the method used in the current study.
This is especially true for female germ cells.