Cell-based screens with CRISPR-Cas9 technology
Cell-based functional genomics screens using CRISPR-Cas9 technology have opened the doors to new discovery in mammalian cell lines. This approach typically relies on use of viral vectors to deliver the sgRNA library. For cell lines for which equivalent viral transduction systems are not available, or for assays in which delivery via viral vectors is problematic, this had been a barrier.
R. Viswanatha and colleagues in the Perrimon lab pushed past this barrier by developing a new approach to pooled-format CRISPR cell screening to Drosophila cells. The method relies on site-specific recombination rather than viral delivery of sgRNAs. One thing we find exicting about this new technology? It is extensible to additional cell types including cells from other insect species.
Seeking new collaborations for mosquito cell projects
The DRSC-BTRR in the Perrimon lab at Harvard Medical School has among its goals to make it possible for laboratories focused on mosquito research to perform genome-wide, pooled format screens in mosquito cell lines. The most approachable screens are 'selections' that identify genes for which knockout confers resistance to a toxin, drug, or other perturbation that normally kills cells or slows division. However, work in other systems suggests that many other screen assays can be performed using this technology, including flow cytometry-based assays.
We are interested to partner with mosquito biologists and others who can further test the screening approach for use in biomedically relevant assays. For some mosquito species, we are actively developing 'screen-ready' cell lines. For others, we can provide reagents that would make it possible for you to first engineer screen-ready cells, then perform CRISPR-based studies including pooled-format screens.
What's available (as of August 2020)
We have developed and tested a screening-ready cell line for the mosquito species Anopheles coluzzii. In addition, the system is being developed for specific cell lines derived from Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus. As noted above, we can empower others to establish the system in additional cell lines by providing relevant plasmid vectors and protocols.
In addition, our bioinformatics group, led by Y. Hu, has developed new online resources relevant to work in mosquitos or mosquito cell lines. One supports ortholog mapping between Drosophila and several mosquito species genes. The other supports identification of pre-computed CRISPR sgRNA designs for multiple mosquito species. Specific Anopheles, Aedes, and Culex species are supported. A YouTube demo video is available here.
In sum, we have available:
- an established platform for CRISPR knockout screening in Anopheles coluzzii cells
- additional modified cell lines and plasmids useful for establishing screening in Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus cell lines
- an online resource for identifying mosquito orthologs of Drosophila genes and vice-versa and a corresponding demo video at YouTube
- a searchable pre-computed database of sgRNA designs (genome view and table view)
Only mosquito cells?
Cultured cell lines are available for many instect and other species. If you can imagine the pooled-format CRISPR screening technology we describe being useful in a cell system you are working with, please feel free to get in touch so we can explore together what might be possible. In addition, we have previously established the approach for Drosophila melanogaster cells.