Efficient Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas

TLDR

CRISPR‑Cas genome engineering in yeast has traditionally required complex plasmid preparations for multiplexed gene knockouts and point mutations. The study demonstrates that co‑transforming a single linearized plasmid with multiple PCR‑generated gRNA and donor DNA cassettes enables high‑efficiency multiplexed integration of point mutations and large constructs. The method uses PCR‑generated gRNA cassettes delivered in any combination to co‑transform a single plasmid, allowing rapid phenotyping of up to five allele combinations and integration of multi‑kilobase pathways across multiple loci in yeast. The approach achieves 64% efficiency for marker‑less triple‑engineering events without selecting for all gRNAs and accelerates complex strain engineering for basic research and industrial fermentation.

Abstract

CRISPR-Cas genome engineering in yeast has relied on preparation of complex expression plasmids for multiplexed gene knockouts and point mutations. Here we show that co-transformation of a single linearized plasmid with multiple PCR-generated guide RNA (gRNA) and donor DNA cassettes facilitates high-efficiency multiplexed integration of point mutations and large constructs. This technique allowed recovery of marker-less triple-engineering events with 64% efficiency without selection for expression of all gRNAs. The gRNA cassettes can be easily made by PCR and delivered in any combination. We employed this method to rapidly phenotype up to five specific allele combinations and identify synergistic effects. To prototype a pathway for the production of muconic acid, we integrated six DNA fragments totaling 24 kb across three loci in naive Saccharomyces cerevisiae in a single transformation. With minor modifications, we integrated a similar pathway in Kluyveromyces lactis. The flexibility afforded by combinatorial gRNA delivery dramatically accelerates complex strain engineering for basic research and industrial fermentation.