title: A Precise Genome Editing Method Reveals Insights into the Activity of Eukaryotic Promoters.
abstract: Despite the availability of whole-genome sequences for almost all model organisms, making faithful predictions of gene expression levels based solely on the corresponding promoter sequences remains a challenge. Plasmid-based approaches and methods involving selection markers are not ideal due to copy-number fluctuations and their disruptive nature. Here, we present a genome editing method using the CRISPR/Cas9 complex and elucidate insights into the activity of canonical promoters in live yeast cells. The method involves the introduction of a novel cut site into a specific genomic location, followed by the integration of an edited sequence into the same location in a scarless manner. Using this method to edit the GAL1 and GAL80 promoter sequences, we found that the relative positioning of promoter elements was critically important for setting promoter activity levels in single cells. The method can be extended to other organisms to decode genotype-phenotype relationships in various gene networks.
authors.affiliation: Department of Molecular Cellular and Developmental Biology, Yale University, 219 Prospect Street, New Haven, CT 06511, USA; Systems Biology Institute, Yale University, 850 West Campus Drive, West Haven, CT 06516, USA.
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Elison
authors.affiliation: Systems Biology Institute, Yale University, 850 West Campus Drive, West Haven, CT 06516, USA; Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, 300 George Street, Suite 501, New Haven, CT 06511, USA.
authors.forename: Ruijie
authors.initials: R
authors.lastname: Song
authors.affiliation: Department of Molecular Cellular and Developmental Biology, Yale University, 219 Prospect Street, New Haven, CT 06511, USA; Systems Biology Institute, Yale University, 850 West Campus Drive, West Haven, CT 06516, USA; Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, 300 George Street, Suite 501, New Haven, CT 06511, USA; Department of Physics, Yale University, 217 Prospect Street, New Haven, CT 06511, USA. Electronic address: murat.acar@yale.edu.
authors.forename: Murat
authors.initials: M
authors.lastname: Acar
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D064113:CRISPR-Cas Systems / Q000235:genetics; D002470:Cell Survival; D000072669:Gene Editing; D016678:Genome; D008954:Models, Biological; D010641:Phenotype; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D029701:Saccharomyces cerevisiae Proteins
keywords: CRISPR; galactose network; genome editing; promoter architecture; yeast