(228a) A Repackaged CRISPR/Cas9 Platform Recasts Non-Homologous End Joining As a Beneficial Instrument in Nonconventional Yeast Engineering

The inability to achieve efficient homology-directed repair (HDR) presents challenges in CRISPR/Cas9-based genome editing in organisms that proficiently employ non-homologous end joining (NHEJ) to fix DNA double-strand breaks (DSBs). Current strategies to combat NHEJ preference involve either knockout/knockdown of NHEJ-associated genes or chemical inhibition of the gene products. In this work, we highlight how the severe reduction in the vitality of NHEJ-deficient strains and the indispensable role of NHEJ in genome maintenance necessitate its preservation to engineer high-performance microbial factories. We illuminate shortcomings associated with the commonly utilized CRISPR platforms in NHEJ-proficient yeast, including 1) a reliance on uptake of multiple DNA fragments via co-transformations, 2) the toxicity of constitutive Cas9 expression, and 3) most importantly, the laborious screening requirements that arise due to the large fractions of false positives in NHEJ-proficient backgrounds. Guided by these insights, we developed a novel CRISPR platform, Lowered Indel Nuclease system Enabling Accurate Repair (LINEAR), which addresses these issues to achieve drastically improved HDR rates (67-100%) compared to those previously reported using traditional CRISPR platforms in four NHEJ-proficient yeasts, demonstrating the cross-species applicability of LINEAR. With our new approach, we demonstrate the utility of NHEJ in strain engineering as a tool for surveying the genomic landscape to identify loci whose spatiotemporal genomic architectures yield favorable expression dynamics of the desired pathway. To elucidate core design principles in engineering non-conventional microbial species, we focus on Scheffersomyces stipitis, the species exhibiting the highest NHEJ proficiency in our survey, and present a case study that alternately employs LINEAR precision editing and NHEJ random integration to rapidly create and optimize a plant-sourced benzylisoquinoline alkaloid production host. Taken together, this work demonstrates how to leverage an antagonizing pair of DNA DSB repairing pathways, synergizing their individual activities to rapidly overcome the challenges in expanding the current collection of microbial cell factories.