TY - JOUR

T1 - CRI-SPA

T2 - a high-throughput method for systematic genetic editing of yeast libraries

AU - Cachera, Paul

AU - Olsson, Helén

AU - Coumou, Hilde

AU - Jensen, Mads L

AU - Sánchez, Benjamín j

AU - Strucko, Tomas

AU - Van den broek, Marcel

AU - Daran, Jean-marc

AU - Jensen, Michael k

AU - Sonnenschein, Nikolaus

AU - Lisby, Michael

AU - Mortensen, Uffe h

PY - 2023

Y1 - 2023

N2 - 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.

AB - 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.

U2 - 10.1093/nar/gkad656

DO - 10.1093/nar/gkad656

M3 - Journal article

C2 - 37572348

VL - 51

JO - Nucleic acids symposium series

JF - Nucleic acids symposium series

SN - 0261-3166

IS - 17

M1 - e91

ER -