ADVANCES IN GENOME EDITING TECHNOLOGIES

抄録

In the five years since the initial demonstration of mammalian genome editing using the Cas9 enzyme, the molecular scissors of the microbial adaptive immune CRISPR system, a number of advancements in genome editing technology have been made with astounding speed. Cas9 has been leveraged for a range of genome manipulation tools, including gene activation and repression as well as modulation of chromatin and DNA modifications. Additional DNA-targeting Cas enzymes have been discovered, broadening the possible targeting space within the human genome and offering greater activity in other species. More recently, RNA-targeting Cas enzymes have been discovered, expanding CRISPR-mediated technologies into the realm of the transcriptome modulation. We have characterized a number of these novel enzymes, known as Cas13, and identified orthologs that work in mammalian cells with high activity and specificity. We have shown that Cas13 can be used to knock down endogenous transcripts as well as serve as a programmable RNA-binding platform. Additionally, we engineered a fusion between Cas13 and the adenine deaminase ADAR to achieve RNA Editing for Precise A-to-I Replacement (REPAIR). We showed that REPAIR has the potential to correct single-base pathogenic mutations at the transcriptional level. REPAIR may be a powerful therapeutic for diseases that affect cell types and tissues not amenable to DNA-based gene therapies, such as neurons and other post-mitotic cells. We are continuing to explore microbial diversity to find new enzymes and systems that can be adapted for use as molecular biology tools and novel therapeutics.