"Discovery and development of CRISPR-associated transposases for next-generation genome engineering"
2023 Advances in Precision Medicine Seminar Series
Speaker: Samuel Sternberg, PhD
Assistant Professor
Department of Biochemistry and Molecular Biophysics
Columbia University
Zoom and In-person: 622 West 168th St. PH-10-405A/B
In recent years, genome editing technologies have advanced from nuclease-based methods using CRISPR-Cas9 that generate DNA double-strand breaks (DSBs), which can cause deleterious side effects, to next-generation approaches that perform controlled chemistry using DSB-independent approaches. Base editing and prime editing are ideally suited for small-scale modifications, but methods to achieve large-payload gene insertion have been lacking. Towards this goal, I will present work from my lab describing a new family of CRISPR-associated transposases (CASTs) that perform highly accurate, targeted integration of DNA payloads via RNA-guided DNA transposition. Unlike conventional CRISPR systems that combine RNA-guided targeting with DNA cleavage, CAST systems exploit nuclease-deficient CRISPR systems for RNA-guided targeting, leading to the site-specific recruitment of transposase enzymes for DNA insertion. We have resolved molecular details of this pathway using a combination of high-throughput sequencing and cryo-electron microscopy, revealing a hierarchical assembly pathway and a structural roadmap to guide engineering efforts. Beyond applying CAST systems as a powerful new tool for bacterial and microbiome engineering, we recently reported our efforts to reconstitute kilobase-scale gene knock-ins in mammalian cells. Our longterm goal is to harness CASTs as a versatile platform technology for integrating therapeutically relevant DNA payloads into the genome to treat human disease.