CRI-SPA: a high-throughput method for systematic genetic editing of yeast libraries

Paul Cachera, Helén Olsson, Hilde Coumou, Mads L. Jensen, Benjamín J. Sánchez, Tomas Strucko, Marcel van den Broek, Jean Marc Daran, Michael K. Jensen, Nikolaus Sonnenschein, Michael Lisby, Uffe H. Mortensen

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Biological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and new research tools allowing simple and rapid mapping of sequence to function are desirable. Here, we describe CRI-SPA, a method allowing the transfer of chromosomal genetic features from a CRI-SPA Donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae. CRI-SPA is based on mating, CRISPR-Cas9-induced gene conversion, and Selective Ploidy Ablation. CRI-SPA can be massively parallelized with automation and can be executed within a week. We demonstrate the power of CRI-SPA by transferring four genes that enable betaxanthin production into each strain of the yeast knockout collection (≈4800 strains). Using this setup, we show that CRI-SPA is highly efficient and reproducible, and even allows marker-free transfer of genetic features. Moreover, we validate a set of CRI-SPA hits by showing that their phenotypes correlate strongly with the phenotypes of the corresponding mutant strains recreated by reverse genetic engineering. Hence, our results provide a genome-wide overview of the genetic requirements for betaxanthin production. We envision that the simplicity, speed, and reliability offered by CRI-SPA will make it a versatile tool to forward systems-level understanding of biological processes.

Original languageEnglish
Article numbere91
Number of pages14
JournalNucleic Acids Research
Issue number17
Publication statusPublished - 2023


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