Analyses of DNA double-strand break repair pathways in tandem arrays of HXT genes of Saccharomyces cerevisiae

Enhancing Homologous Recombination Efficiency in Pichia pastoris for Multiplex Genome Integration Using Short Homology Arms

“…Aiming to enhance HR efficiency in P. pastoris , we introduced the recombination machinery from S. cerevisiae (Figure ). Thus, core genes related to HR, including RAD52 , RAD59 , , MRE11 , , and SAE2 , were overexpressed under strong constitutive promoters (i.e., pGAP and pTEF1 ) and inserted into the chromosome of P. pastoris , whose effects on genome integration efficiency were evaluated using reporter gene expression cassettes. We chose RAD52 , a key HR-related gene, as the first target to be investigated.…”

“…In the present study, we aim to achieve multiplex genome integration using short (i.e., ∼40 bp) homology arms in P. pastoris , by introducing HR machinery from S. cerevisiae . First, we evaluated the effects of overexpressing S. cerevisiae HR related genes (Figure ), including RAD52 , RAD59 , , MRE11 , , and SAE2 , on genome integration efficiency as well as cell fitness. Then, we constructed P. pastoris strains (Supplementary Table S1) with enhanced HR efficiency to achieve efficient single-, two-, and three-loci genome integration using ∼40 bp homology arms.…”

A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris

“…In S. cerevisiae , after the DSB is recognized, the single-stranded DNA is generated by SAE2 [ 35 ]. Afterwards, the strand invasion and exchange are accomplished by a series of proteins from RAD family, including RAD51 , RAD52 [ 36 ], RAD59 [ 37 ]. Furthermore, the much higher positive rates can be obtained by overexpressing endogenous PpRAD52 in P. pastoris [ 9 ].…”