Developing GDi-CRISPR System for Multi-copy Integration in Saccharomyces cerevisiae

In recent years, Saccharomyces cerevisiae has been widely used in the production of biofuels and value-added chemicals. To stably express the target products, it is necessary to integrate multiple target genes into the chromosome of S. cerevisiae. CRISPR multi-copy integration technology relying on delta sites has been developed, but it often requires the help of high-throughput screening or resistance markers, resulting in non-replicability and high cost. This study aims to develop a low-cost and easy-to-use multi-copy integration tool in S. cerevisiae. Firstly, twenty-one Cas proteins from different microorganisms were tested in S. cerevisiae to find the functional Cas proteins with optimal cleavage ability. Results showed that eight Cas proteins can complete gene editing. However, most of the transformants have low copy numbers, which may be caused by high cutting efficiency exceeding the repair rate. Therefore, the effect of donor translocation order was further investigated. Results showed that 4 copies were obtained by donor first translocation. Then, the gene drive delta site integration system by the CRISPR system (GDi-CRISPR) was developed by combining gene drive principle and CRISPR system. To be clear, the gRNA was put into donor fragments. Then, both of them were integrated into the genome, which can drive further cutting and repair due to increasing number of gRNA. Instead of high-throughput screening or resistance pressure, 6 copies were obtained in only 5-6 days using the GDi-CRISPR system. It is expected to further advance the development of S. cerevisiae multi-copy integration tools.