Stably Integrating an Inducible CRISPR-Cas9 to Aug- ment the Mammalian Immune System to Protect Against Viral Infections

Vaccines are the best preventative measure available to reduce the sever-<br> ity of a viral infection. However, a vaccine’s dependency on the immune system<br> limits its efficacy in immunocompromised patients. This work explores how the bac-<br> terial CRISPR-Cas9 system can be repurposed to protect against viral infections in<br> human cells. The A549 lung adenocarcinoma cell line was genetically modified to<br> stably express a doxycycline-inducible Cas9 and a guide RNA that targeted a lu-<br> ciferase reporter plasmid driven by the immediate-early cytomegalovirus promoter.<br> This luciferase reporter was a marker for viral infection. Six guide RNA sequences<br> were tested, and it was found that luciferase expression decreased by up to 98 per-<br> cent when Cas9 was active relative to a non-targeting control. This indicated Cas9<br> cleaved the transfected plasmid and effectively protected the cell from foreign DNA<br> expression. Further testing with qPCR identified that four to fourteen copies of the<br> Cas9 gene and two to ten copies of the guide RNA were present in the genome<br> of the A549 cells, validating that both successfully integrated. To address Cas9’s<br> high specificity, viral regions that are unlikely to be mutation tolerant were identi-<br> fied through the MAFFT alignment of thirteen cytomegalovirus genomes. This work<br> is novel because it characterizes an inducible Cas9 system in human cells and<br> shows its efficacy against double-stranded DNA-based cytomegaloviruses, which<br> are widely prevalent and have no cure. Overall, CRISPR systems have the po-<br> tential to revolutionize the way we protect immunocompromised patients from viral<br> infections