Sequencing and assembly of engineered strains of Scheffersomyces stipitis. Scheffersomyces stipitis

In CRISPR-Cas9 systems, It is a challenge to achieve effective homology-directed repair (HDR) 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 research, 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 developed a novel CRISPR platform (Lowered Indel Nuclease system Enabling Accurate Repair (LINEAR)), which drastically improved HDR rates (67-100%) compared to those previously reported using traditional CRISPR platforms in four NHEJ-proficient yeasts