iSeq 2.0: A Modular and Interchangeable Toolkit for Interaction Screening in Yeast.

We developed a flexible toolkit for combinatorial screening in Saccharomyces cerevisiae, which generates large libraries of cells, each uniquely barcoded to mark a combination of DNA elements. This interaction sequencing platform (iSeq 2.0) includes genomic landing pads that assemble combinations through sequential integration of plasmids or yeast mating, 15 barcoded plasmid libraries containing split selectable markers (URA3AI, KanMXAI, HphMXAI, and NatMXAI), and an array of ∼24,000 "double-barcoder" strains that can make existing yeast libraries iSeq compatible. Various DNA elements are compatible with iSeq: DNA introduced on integrating plasmids, engineered genomic modifications, or entire genetic backgrounds. DNA element libraries are modular and interchangeable, and any two libraries can be combined, making iSeq capable of performing many new combinatorial screens by short-read sequencing.

[1]  Ronald W. Davis,et al.  Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. , 1999, Science.

[2]  R. Schiestl,et al.  High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method , 2007, Nature Protocols.

[3]  Sean R. Collins,et al.  A comprehensive strategy enabling high-resolution functional analysis of the yeast genome , 2008, Nature Methods.

[4]  Robert P. Davey,et al.  Population genomics of domestic and wild yeasts , 2008, Nature.

[5]  A. Barabasi,et al.  High-Quality Binary Protein Interaction Map of the Yeast Interactome Network , 2008, Science.

[6]  Sarah Barber,et al.  Sequencing and analysis of 10,967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis reveals post-tetraploidization transcriptome remodeling. , 2006, Genome research.

[7]  Sean R. Collins,et al.  Conservation and Rewiring of Functional Modules Revealed by an Epistasis Map in Fission Yeast , 2008, Science.

[8]  Sourav Bandyopadhyay,et al.  Rewiring of Genetic Networks in Response to DNA Damage , 2010, Science.

[9]  T. Hughes,et al.  Exploration of Essential Gene Functions via Titratable Promoter Alleles , 2004, Cell.

[10]  R. Schiestl,et al.  Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method , 2007, Nature Protocols.

[11]  Ben Lehner,et al.  The genetic landscape of a physical interaction , 2018, eLife.

[12]  M. Dante,et al.  Multifunctional yeast high-copy-number shuttle vectors. , 1992, Gene.

[13]  Bridget E. Begg,et al.  A Proteome-Scale Map of the Human Interactome Network , 2014, Cell.

[14]  C. Myers,et al.  Synthetic genetic array (SGA) analysis in Saccharomyces cerevisiae and Schizosaccharomyces pombe. , 2010, Methods in enzymology.

[15]  Ulrich Schlecht,et al.  A scalable double-barcode sequencing platform for characterization of dynamic protein-protein interactions , 2017, Nature Communications.

[16]  H. Albert,et al.  Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. , 1995, The Plant journal : for cell and molecular biology.

[17]  Sasha F. Levy,et al.  A method for high‐throughput production of sequence‐verified DNA libraries and strain collections , 2017, Molecular systems biology.

[18]  Timothy K Lu,et al.  Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM , 2016, Proceedings of the National Academy of Sciences.

[19]  Gary D Bader,et al.  Global Mapping of the Yeast Genetic Interaction Network , 2004, Science.

[20]  R. Ranganathan,et al.  Origins of Allostery and Evolvability in Proteins: A Case Study , 2016, Cell.

[21]  Y. Zhang,et al.  Saccharomyces cerevisiae ATM orthologue suppresses break-induced chromosome translocations , 2008, Nature.

[22]  Sasha F. Levy,et al.  Unbiased Fitness Estimation of Pooled Barcode or Amplicon Sequencing Studies. , 2018, Cell systems.

[23]  T. Badea,et al.  Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies , 2015, G3: Genes, Genomes, Genetics.

[24]  James R. Knight,et al.  A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae , 2000, Nature.

[25]  M. Laub,et al.  Evolving New Protein-Protein Interaction Specificity through Promiscuous Intermediates , 2015, Cell.

[26]  E. O’Shea,et al.  Global analysis of protein localization in budding yeast , 2003, Nature.

[27]  E. Levy,et al.  Genome-wide C-SWAT library for high-throughput yeast genome tagging , 2018, Nature Methods.

[28]  C. Landry,et al.  An in Vivo Map of the Yeast Protein Interactome , 2008, Science.

[29]  J. Mccusker,et al.  Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae , 1999, Yeast.

[30]  R. Ozawa,et al.  A comprehensive two-hybrid analysis to explore the yeast protein interactome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[31]  I. Saito,et al.  Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. , 1998, Gene.

[32]  Weiping Ma,et al.  Embryogenesis Microarray for Profiling Gene Expression Patterns during 15,000 Unique Zebrafish Est Clusters and Their Future Use in Material Supplemental , 2022 .

[33]  Sasha F. Levy,et al.  iSeq: A New Double-Barcode Method for Detecting Dynamic Genetic Interactions in Yeast , 2016, G3: Genes, Genomes, Genetics.

[34]  J. Boeke,et al.  A useful colony colour phenotype associated with the yeast selectable/counter‐selectable marker MET15 , 1996, Yeast.

[35]  T. Schlake,et al.  Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. , 1994, Biochemistry.

[36]  David E Hill,et al.  Pooled‐matrix protein interaction screens using Barcode Fusion Genetics , 2016, Molecular systems biology.

[37]  Aaron N. Chang,et al.  Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions , 2017, Nature Methods.

[38]  Thomas M Green,et al.  A public genome-scale lentiviral expression library of human ORFs , 2011, Nature Methods.

[39]  David Baker,et al.  High-throughput characterization of protein–protein interactions by reprogramming yeast mating , 2017, Proceedings of the National Academy of Sciences.

[40]  M. Jayaram,et al.  Site-specific recombinase, R, encoded by yeast plasmid pSR1. , 1992, Journal of molecular biology.

[41]  Grant W. Brown,et al.  Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map , 2007, Nature.

[42]  D. Garfinkel,et al.  Single-step selection for Ty1 element retrotransposition. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Gavin Sherlock,et al.  Quantitative evolutionary dynamics using high-resolution lineage tracking , 2015, Nature.

[44]  Gary D Bader,et al.  The Genetic Landscape of a Cell , 2010, Science.

[45]  D. Stillman,et al.  New ‘marker swap’ plasmids for converting selectable markers on budding yeast gene disruptions and plasmids , 2003, Yeast.

[46]  D. Reich,et al.  Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture , 2012, Genome research.

[47]  Adam Frost,et al.  Functional Repurposing Revealed by Comparing S. pombe and S. cerevisiae Genetic Interactions , 2012, Cell.

[48]  Grant W. Brown,et al.  Mapping DNA damage‐dependent genetic interactions in yeast via party mating and barcode fusion genetics , 2017, bioRxiv.

[49]  D. Gottschling,et al.  The Mother Enrichment Program: A Genetic System for Facile Replicative Life Span Analysis in Saccharomyces cerevisiae , 2009, Genetics.

[50]  J. Boeke,et al.  Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR‐mediated gene disruption and other applications , 1998, Yeast.

[51]  D. G. Wang,et al.  Solid-phase reversible immobilization for the isolation of PCR products. , 1995, Nucleic acids research.

[52]  Beat Lutz,et al.  Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein. , 2002, Nucleic acids research.

[53]  Gaelen T. Hess,et al.  Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions , 2017, Nature Biotechnology.

[54]  Adam P. Rosebrock,et al.  A global genetic interaction network maps a wiring diagram of cellular function , 2016, Science.

[55]  Ronald W. Davis,et al.  Functional profiling of the Saccharomyces cerevisiae genome , 2002, Nature.

[56]  F. Nagawa,et al.  Control of gene expression by artificial introns in Saccharomyces cerevisiae. , 1989, Science.

[57]  Lu Zhao,et al.  Bartender: a fast and accurate clustering algorithm to count barcode reads , 2018, Bioinform..

[58]  Nevan J Krogan,et al.  The next frontier of systems biology: higher-order and interspecies interactions , 2010, Genome Biology.

[59]  Gilles Fischer,et al.  bz-rates: A Web Tool to Estimate Mutation Rates from Fluctuation Analysis , 2015, G3: Genes, Genomes, Genetics.

[60]  Kei-Hoi Cheung,et al.  Large-scale analysis of the yeast genome by transposon tagging and gene disruption , 1999, Nature.

[61]  Timothy K Lu,et al.  Massively parallel high-order combinatorial genetics in human cells , 2015, Nature Biotechnology.

[62]  Sundari Suresh,et al.  Quantitative analysis of protein interaction network dynamics in yeast , 2017, Molecular systems biology.