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

疫苗是降低病毒感染严重性的最佳预防措施。然而，疫苗对免疫系统的依赖性限制了其在免疫缺陷患者中的有效性。本研究探讨了如何将细菌CRISPR-Cas9系统重新用于保护人类细胞免受病毒感染。A549肺腺癌细胞系被基因改造，以稳定表达由即早型巨细胞病毒启动子驱动的doxycycline诱导型Cas9和靶向荧光素酶报告质粒的引导RNA。该荧光素酶报告质粒是病毒感染的标志物。测试了六种引导RNA序列，发现与非靶向对照组相比，当Cas9活性时，荧光素酶表达量降低了高达98%。这表明Cas9切割了转染的质粒，并有效保护细胞免受外来DNA表达。进一步的qPCR测试发现，A549细胞基因组中存在四到十四个Cas9基因拷贝和两个到十个引导RNA拷贝，验证了它们均成功整合。为了解决Cas9的高特异性，通过MAFFT对十三种巨细胞病毒基因组进行比对，确定了不太可能发生突变的病毒区域。这项工作具有创新性，因为它在人类细胞中描述了一种可诱导的Cas9系统，并展示了其对基于双链DNA的巨细胞病毒的疗效，这些病毒广泛流行且无治愈方法。总的来说，CRISPR系统有潜力彻底改变我们保护免疫缺陷患者免受病毒感染的方式。