CRISPR/Cas9 Module Project

     Given recent advancements in the application of the CRISPR/Cas9 system to modifying organisms, and its obvious utility in the field of biological engineering, I believe a useful future module that can be developed for 20.109 could involve applying Cas9.
     My experience with Cas9 has been with using it to insert fluorescent tags endogenously into C. elegans. Soon, I will be using it to create point mutations in specific genes in the nematode in order to potentially create analogs for certain human genetic disorders. I can easily imagine applying my experience with this technology towards developing a project that could be done within the time-span of a module. Working with nematodes would not be likely though, since current methods of genome editing in these organisms requires microinjection skills, which can be tricky to learn. However, this technology can be used with models that students already work with in 20.109: mouse embryonic stem cells.
     A possible project could simply be a modified version of this semester's first project. Students could still analyze recombination of EGFP-coding plasmids, however, they could also study how this recombination is affected when certain genes in the ES cells are knocked down by CRISPR. Knocking out a gene is relatively easy to perform with CRISPR, and would require the fabrication of only one more plasmid per gene that is to be knocked down. Given the right guide RNA, knock downs would be guaranteed given adequate lipofections, and engineered strains of ES cells could be studied with the recombination assay already in use. So that the final assignment does not become too complex, the experimental variation can shift from changing the quantity and type of damaged DNA lipofected, to how certain strains of ES cells accomplish homologous recombination given deletions in certain genes. Such a project would provide students with all the lab experience they currently obtain in the module as it is now, and also instill in them knowledge and experience with a technology that will likely be useful in their future.