Speaker: Krishanu Saha, assistant professor in the University of Wisconsin College of Engineering with biomedical engineering as his area of expertise

Abstract: Genome surgery frequently involves coordinated action among many different types of biomolecules (proteins, RNAs and DNAs), and assembling these biomolecules appropriately at the genomic site of surgery provides an opportunity to increase the efficiency and precision of genome editing. We hypothesized that co-delivery of the CRISPR machinery within a single nanoparticle could help generate more predictable editing outcomes. We developed a modular RNA aptamer-streptavidin strategy, termed S1mplex, to complex CRISPR-Cas9 ribonucleoproteins (RNPs) with a nucleic acid donor template (Carlson-Stevermer, Nature Communications 2017). The co-localization of the donor template with the Cas9 nuclease is likely a key reason why we achieve up to 4 precise edits for every imprecise edit (7-18 fold higher than standard methods). The S1mplex technique can also be used to assemble new molecular cargoes with the Cas9 RNP. One principle for cell engineering arising from this work, which my lab and several other labs now routinely follow, is avoiding imprecise editing outcomes when fixing short mutations (one to ten bases) by co-delivery of all the components of the genome editing machinery. These advances with versatile, preassembled reagents could greatly reduce the time and cost of in vitro/ex vivo gene editing applications and aid in the development of increased and serial dosing regimens for somatic cell gene editing in vivo. We also noted that transfection agents do not uniformly encapsulate proper combinations of the CRISPR-Cas9 machinery. Through collaborative projects, we have now created novel non-viral polymeric delivery strategies for RNP assemblies. Two different strategies have been developed: an artificial polymer vesicle (Wang, ACS Applied Interfaces 2018) and cell-degradable polymer coating. I will describe how we are currently using these reagents to study gene editing in cells/tissues derived from patient-derived pluripotent stem cells and in vivo within transgenic animals.

Hosted by: Molecular, Cellular and Developmental Biology Graduate Student Organization