ERASR: Epigenetic Remodeling of APOBEC and Selective RNA Editing

PI:

Valter Tucci

Email:

valter.tucci@iit.it

Affiliation:

Istituto italiano di tecnologia (IIT)

ORCID:

0000-0002-3796-3826

In recent years we witnessed a fast revolution in gene-editing technologies, such as clustered regularly interspaced palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. CRISPR technology is based on DNA breaks and homology-directed repair mechanisms, which can be programmed by noncoding single guide RNAs (sgRNAs) to guide specific nuclease (e.g., Cas9 and all successively modified proteins) to induce site-specific DNA cleavage in effectively any cellular system and organism. Despite the potential of DNA editing in gene therapy, single-strand nicks bring significant cellular toxicity, currently preventing the large use of CRISPR-based applications in gene therapy. The relatively low efficiency of precise homology-directed repairing, the presence of off-target mutations, and the difficulties of introducing CRISPR nucleases and targeting templates into stem cells are common issues in exploiting CRISPR gene-editing to treat human diseases. To overcome these issues, it has been proposed to adapt CRISPR technology to control the epigenetic regulatory mechanisms of gene transcription. Among them, DNA methylation on CpG dinucleotides regulates the opening or the closure of chromatin, binding specific transcription factors to the gene promoter and assembling the transcription machinery. Therefore, we developed a novel Cas-based Targeting Units for Specific Epigenetic Remodelling (TUSER), that can precisely and timely regulate the expression of target mammalian genes by rewriting their epigenetic landscape without introducing changes to the underlying DNA sequences. This too can be potentially applied to treat any gene expression aberration. Among the CN3 – Spoke 6, we put our experience at service of the RNA drugs development. In particular, our tool can be instrumental in modifying the gene expression frame or the cellular environment that reduces the efficacy of RNA-based technology. One example consists in our collaboration with the CNR (Consiglio Nazionale delle Ricerche) group of the task 6.1.1, to modulate physiological mRNA editing enzymes as a cancer treatment. In particular, we modified our technology to include both the Cas guide and APOBEC scaffold sequence as intron of the epigenetic writer. This strategy consent to epigenetically increase the expression of APOBEC in the cancer cells and parallely produces the guide for the RNA editing of this protein against specific oncogenic targets.

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