title: Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design.
abstract: BACKGROUND
  Genome-scale CRISPR interference (CRISPRi) has been used in human cell lines; however, the features of effective guide RNAs (gRNAs) in different organisms have not been well characterized. Here, we define rules that determine gRNA effectiveness for transcriptional repression in Saccharomyces cerevisiae.
  
  
  RESULTS
  We create an inducible single plasmid CRISPRi system for gene repression in yeast, and use it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identifies previously described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments using gRNA libraries allows us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility are clearly more effective. We also find that the best region to target gRNAs is between the transcription start site (TSS) and 200 bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity), as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets.
  
  
  CONCLUSIONS
  Our results establish a powerful functional and chemical genomics screening method and provide guidelines for designing effective gRNAs, which consider chromatin state and position relative to the target gene TSS. These findings will enable effective library design and genome-wide programmable gene repression in many genetic backgrounds.
authors.affiliation: Stanford Genome Technology Center, Department of Biochemistry, Stanford University, 3165 Porter Drive, Palo Alto, CA, 94304, USA.|Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Justin D
authors.initials: JD
authors.lastname: Smith
authors.affiliation: Stanford Genome Technology Center, Department of Biochemistry, Stanford University, 3165 Porter Drive, Palo Alto, CA, 94304, USA.
authors.forename: Sundari
authors.initials: S
authors.lastname: Suresh
authors.affiliation: Stanford Genome Technology Center, Department of Biochemistry, Stanford University, 3165 Porter Drive, Palo Alto, CA, 94304, USA.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schlecht
authors.affiliation: Department of Anesthesia, Stanford University School of Medicine, Stanford University, Stanford, California, 94305, USA.
authors.forename: Manhong
authors.initials: M
authors.lastname: Wu
authors.affiliation: European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Genome Campus, Hinxton, CB101SD, UK.
authors.forename: Omar
authors.initials: O
authors.lastname: Wagih
authors.affiliation: Department of Anesthesia, Stanford University School of Medicine, Stanford University, Stanford, California, 94305, USA.
authors.forename: Gary
authors.initials: G
authors.lastname: Peltz
authors.affiliation: Stanford Genome Technology Center, Department of Biochemistry, Stanford University, 3165 Porter Drive, Palo Alto, CA, 94304, USA.
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
authors.affiliation: Stanford Genome Technology Center, Department of Biochemistry, Stanford University, 3165 Porter Drive, Palo Alto, CA, 94304, USA.|Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.|European Molecular Biology Laboratory (EMBL), Genome Biology Unit, 69117, Heidelberg, Germany.
authors.forename: Lars M
authors.initials: LM
authors.lastname: Steinmetz
authors.affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, California, USA. leopold.parts@sanger.ac.uk.|European Molecular Biology Laboratory (EMBL), Genome Biology Unit, 69117, Heidelberg, Germany. leopold.parts@sanger.ac.uk.|Current address: Wellcome Trust Sanger Institute, Hinxton, CB101SA, UK. leopold.parts@sanger.ac.uk.
authors.forename: Leopold
authors.initials: L
authors.lastname: Parts
authors.affiliation: Stanford Genome Technology Center, Department of Biochemistry, Stanford University, 3165 Porter Drive, Palo Alto, CA, 94304, USA. bstonge@stanford.edu.
authors.forename: Robert P
authors.initials: RP
authors.lastname: St Onge
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002843:Chromatin / Q000235:genetics; D016681:Genome, Fungal; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics; D009707:Nucleosomes / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D024363:Transcription Initiation Site
chemical_list: D002843:Chromatin; D009707:Nucleosomes; D000094704:RNA, Guide, CRISPR-Cas Systems; D029701:Saccharomyces cerevisiae Proteins; D006899:Mixed Function Oxygenases; C547779:ERG25 protein, S cerevisiae