LOBSTERS: a modular vector series with a diverse set of Cas nucleases and plasmid selection markers for multiplex genome editing in Saccharomyces cerevisiae

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Abstract The budding yeast Saccharomyces cerevisiae is a central model organism in genetics and synthetic biology, yet efficient multiplex genome editing remains difficult because many toolkits are restricted by limited plasmid selection markers and reduced efficiency when targeting multiple loci. In our previous work, we introduced a CRISPR-based system incorporating three nucleases with distinct PAM specificities but it was available only with the URA3 marker. Here we present LOBSTERS, an expanded modular vector series that retains the PAM-diverse nucleases (SpCas9, SaCas9, and enAsCas12a) while extending marker options to seven. This design enables the simultaneous use of multiple plasmids in one transformation, supporting scalable and flexible genome editing. Proof-of-concept experiments demonstrated efficient ADE2 and ADE3 deletions with colorimetric readouts, coordinated tagging of essential proteins (Cdc3 and Cse4) without compromising function, and recapitulation of three quantitative trait variants ( RME1, TAO3 , and MKT1 ) underlying sporulation efficiency. Together these results establish LOBSTERS as a robust and versatile platform for multiplex genome editing in S. cerevisiae . By enabling coordinated modification of essential proteins, genetic interactions, and quantitative trait variants, LOBSTERS provides a broadly applicable resource for functional cell biology and synthetic biology in yeast. Significance Statement LOBSTERS integrates three PAM-diverse nucleases with seven plasmid selection markers, overcoming the single-marker limitation of previous yeast genome editing toolkits. The system enables efficient simultaneous editing at multiple loci, demonstrated by functional tagging of essential proteins and recapitulation of quantitative trait variants. By broadening the editable space and lowering barriers to complex genotype construction, LOBSTERS provides a widely applicable resource for yeast cell biology and synthetic biology.

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