(627g) Invited Talk: New Tools for High Throughput, Multiplexable Transcriptional Control and for In Vivo Targeted Mutations

We’ll detail our recent efforts to develop two new systems and synthetic biology tools, as well as describe their application in the model yeast, Saccharomyces cerevisiae. First, we’ll describe how we optimized a modular method to enable large-scale transcriptional control of several genes at once in individual cells. This method takes advantage of an optimized overlap-extension PCR-based process to overcome difficulties associated with construction of ‘gRNA’ arrays (i.e., multi-sgRNA expression cassettes), is applicable at the library scale, and is an ideal tool for study of complex cellular phenotypes. Secondly, we’ll describe our development of a high-activity yeast diversifying base editor, or yDBE. Beginning with an initial standard ‘base editor architecture’ that uses dCas9 and scaffolded gRNAs to recruit a mutagenizing cytidine deaminase enzyme to a specific DNA locus, we’ll show how we improved both deaminase enzyme activity and scaffolded gRNA design to greatly enhance the yDBE’s mutational rate. We further detail the ‘mutational window’ afforded by our yDBE, show that it is compatible with our efforts to target multiple DNA loci in single cells, and demonstrate its utility by using it to enhance the affinity of a well-characterized surface-displayed scFv and to increase the strength of yeast promoter.