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.
UR - http://www.scopus.com/inward/record.url?scp=85172424180&partnerID=8YFLogxK
U2 - 10.1093/nar/gkad656
DO - 10.1093/nar/gkad656
M3 - Article
C2 - 37572348
AN - SCOPUS:85172424180
SN - 0305-1048
VL - 51
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 17
M1 - e91
ER -