Clearing of Foreign Episomal DNA from Human Cells by CRISPRa-Mediated Activation of Cytidine Deaminases

Abstract

Restriction of foreign DNA is a fundamental defense mechanism required for maintaining genomic stability and proper function of mammalian cells. APOBEC cytidine deaminases are crucial effector molecules involved in clearing pathogenic DNA of viruses and other microorganisms and improperly localized self-DNA (DNA leakages). Mastering the expression of APOBEC provides the crucial means both for developing novel therapeutic approaches for combating infectious and non-infectious diseases and for numerous research purposes. In this study, we report successful application of a CRISPRa approach to effectively and specifically overexpress APOBEC3A and APOBEC3B deaminases and describe their effects on episomal and integrated foreign DNA. This method increased target gene transcription by >6–50-fold in HEK293T cells. Furthermore, CRISPRa-mediated activation of APOBEC3A/APOBEC3B suppressed episomal but not integrated foreign DNA. Episomal GC-rich DNA was rapidly destabilized and destroyed by CRISPRa-induced APOBEC3A/APOBEC3B, while the remaining DNA templates harbored frequent deaminated nucleotides. To conclude, the CRISPRa approach could be readily utilized for manipulating innate immunity and investigating the effects of the key effector molecules on foreign nucleic acids.

Immunity and Viral Infections: Modulating Antiviral Response via CRISPR–Cas Systems

“…Similarly, overexpressed AID efficiently deaminates HBV cccDNA and purges viral DNA out from the nuclei of infected cells [ 253 ]. It should be noted that the activation of APOBEC3 factors by dCas-based approaches efficiently degrades episome-like genomes that are natural targets of APOBEC proteins but does not efficiently deaminate viral genomes integrated into human chromosomes [ 254 ]. Transient gene activation is an important advantage of CRISPRa, as prolonged overexpression of host restriction factors can induce pathologic states, including toxicity and malignant transformation [ 255 ].…”

CRISPR-Cas systems for diagnosing infectious diseases

“…Using genetic engineering, CRISPR-Cas systems have been adapted for use in humans and are now being modified and enhanced at an extraordinary pace, enabling precise editing of virtually any DNA or RNA molecule in the body. CRISPR-Cas-based approaches are being tested to treat hereditary, infectious, and other diseases, as well as in methods of molecular visualization and other applications [14] , [15] , [16] .…”