CILF: CRISPR/Cas9 based integration of large DNA fragments in Saccharomyces cerevisiae

Abstract Genome integration technology has markedly expedited the construction of cell factories. However, its application is currently limited by the inefficient integration of large DNA fragments. Here, we report a CRISPR/Cas9 based integration of large DNA fragments (CILF) method to efficiently integrate large DNA fragments in Saccharomyces cerevisiae . In this approach, a fusion protein, Cas9‐Brex27‐FadR, was employed for the targeted delivery of donor plasmid to double‐strand breaks (DSBs), while simultaneously recruiting Rad51 to enhance the efficiency of homologous recombination (HR). Our findings demonstrate that this method can achieve an integration efficiency of 98% for 10 kb DNA fragments and nearly 80% for 40 kb DNA fragments at a single site, using donor plasmids with 1000 bp homology arms (HAs) and 12 FadR binding sites (BSs). The CILF technique significantly enriches the synthetic biology toolbox of S. cerevisiae , offering significant potential to propel advancements in both synthetic biology and metabolic engineering.