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-

ity of a viral infection. However, a vaccine’s dependency on the immune system

limits its efficacy in immunocompromised patients. This work explores how the bac-

terial CRISPR-Cas9 system can be repurposed to protect against viral infections in

human cells. The A549 lung adenocarcinoma cell line was genetically modified to

stably express a doxycycline-inducible Cas9 and a guide RNA that targeted a lu-

ciferase reporter plasmid driven by the immediate-early cytomegalovirus promoter.

This luciferase reporter was a marker for viral infection. Six guide RNA sequences

were tested, and it was found that luciferase expression decreased by up to 98 per-

cent when Cas9 was active relative to a non-targeting control. This indicated Cas9

cleaved the transfected plasmid and effectively protected the cell from foreign DNA

expression. Further testing with qPCR identified that four to fourteen copies of the

Cas9 gene and two to ten copies of the guide RNA were present in the genome

of the A549 cells, validating that both successfully integrated. To address Cas9’s

high specificity, viral regions that are unlikely to be mutation tolerant were identi-

fied through the MAFFT alignment of thirteen cytomegalovirus genomes. This work

is novel because it characterizes an inducible Cas9 system in human cells and

shows its efficacy against double-stranded DNA-based cytomegaloviruses, which

are widely prevalent and have no cure. Overall, CRISPR systems have the po-

tential to revolutionize the way we protect immunocompromised patients from viral

infections