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*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

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
JournalNucleic Acids Research
Volume51
Issue number17
Number of pages14
ISSN0305-1048
DOIs
Publication statusPublished - 2023

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