(4fa) A Repackaged CRISPR/Cas9 Platform Recasts Non-Homologous End Joining As a Beneficial Instrument in Nonconventional Yeast Engineering

Inefficient homology-directed repair (HDR) constrains CRISPR/Cas9 genome editing in organisms that preferentially employ non-homologous end joining (NHEJ) to fix DNA double-strand breaks (DSBs). Current strategies to alleviate NHEJ proficiency involve NHEJ disruption. To confer precision editing without NHEJ disruption, we identified shortcomings of conventional CRISPR platforms and developed a novel CRISPR platform, Lowered Indel Nuclease system Enabling Accurate Repair (LINEAR), which drastically enhanced HDR rates (67-100%) compared to previous reports using conventional platforms in four NHEJ-proficient yeasts. With NHEJ preserved, we demonstrate its ability to survey genomic landscapes, identifying loci whose spatiotemporal genomic architectures yield favorable expression dynamics for heterologous pathways. We present a case study that deploys LINEAR precision editing and NHEJ-mediated random integration to rapidly engineer and optimize a microbial factory to produce (S)-norcoclaurine. Taken together, this work demonstrates how to leverage an antagonizing pair of DNA DSB repair pathways to expand the current collection of microbial factories.