Chakravarti, R. | bioRxiv Science

bioRxiv2025

A genetic screen to identify deubiquitinases as regulators of IRF7

Virus infection rapidly induces the production and secretion of interferons (IFNs), amplifying antiviral responses in infected and neighboring uninfected cells. IFN regulatory factor 7 (IRF7), the master transcription factor, is pivotal in IFN induction, particularly in myeloid cells. Ubiquitination of IRF7 is essential for its transcriptional activation; however, the underlying molecular mechanisms remain poorly understood. We hypothesized that deubiquitinases (DUBs) act as endogenous regulators of IRF7 activity and conducted a genetic screen using a human DUB-targeted siRNA library. This screen identified USP2 as a positive regulator and OTUD5 as a negative regulator of IRF7 activity. OTUD5, an inducible DUB, physically interacted with IRF7 and inhibited its K63-linked ubiquitination, thereby suppressing IRF7 activation. Conversely, USP2 promoted IRF7 activity by binding to IRF7 and removing K27-linked ubiquitin chains, which we found to be inhibitory. Specifically, K27-linked ubiquitination impeded phosphorylation of IRF7, a critical step for its activation. Collectively, our genetic screen and mechanistic studies uncovered USP2 and OTUD5 as novel modulators of IRF7 function, providing new insights into the regulation of antiviral immunity.

immunology↗

bioRxiv2023

A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9

CRISPR-Cas9 is the newest technology available for targeted genome editing. It is very efficient and cheap compared to other genome editing techniques. However, its therapeutic application is limited due to its off-target activity. To have a better understanding of this off-target effect, we concentrated our efforts on its mismatch-prone PAM distal end. Current off-target prediction algorithms use RNA-DNA complementation derived energy as a major factor in predicting off-target effect. RNA-DNA complementation derived energy drives Cas9 conformational change, which in turn drives its functional activity. In the case of lower RNA-DNA complementarity, a partial conformational change occurs resulting in a slower reaction rate and partial activity. However, extensive mismatches are often tolerated despite lower complementation derived energy available from RNA: DNA duplex formation. Thus, the off-target activity of Cas9 depends directly on the nature of mismatches which in turn result in deviation of the active site of the enzyme due to structural instability in the duplex strand. In order to test the hypothesis, we have designed an array of mismatched target sites and performed in vitro and cell line-based experiments to assess the effects of PAM distal mismatches in Cas9 activity. For further mechanistic validation, Molecular dynamics simulation was performed and it revealed that certain mismatch mutations induced pronounced conformational instability within the RNA-DNA duplex, leading to elevated root mean square deviation (RMSD) values. We found that, target sites having mismatches in the 18th to 16th position upstream of the PAM showed no to little activity.

genomics↗