Multiplexed precision genome editing with trackable genomic barcodes in yeast

A method to introduce defined mutations into the yeast genome enables saturation mutagenesis of a gene and genome-scale introduction of genetic variants. Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess the phenotypic...

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Bibliographic Details
Published in:	Nature biotechnology Vol. 36; no. 6; pp. 512 - 520
Main Authors:	Roy, Kevin R, Smith, Justin D, Vonesch, Sibylle C, Lin, Gen, Tu, Chelsea Szu, Lederer, Alex R, Chu, Angela, Suresh, Sundari, Nguyen, Michelle, Horecka, Joe, Tripathi, Ashutosh, Burnett, Wallace T, Morgan, Maddison A, Schulz, Julia, Orsley, Kevin M, Wei, Wu, Aiyar, Raeka S, Davis, Ronald W, Bankaitis, Vytas A, Haber, James E, Salit, Marc L, St.Onge, Robert P, Steinmetz, Lars M
Format:	Journal Article
Language:	English
Published:	New York Nature Publishing Group US 01-07-2018 Nature Publishing Group
Subjects:	45 45/41 45/70 49 631/1647 631/1647/1513/1967/3196 631/208 631/208/191/1908 631/553 Agriculture Amino Acid Substitution Amino acids Baking yeast Bar codes Bioinformatics Biomedical Engineering/Biotechnology Biomedicine Biotechnology CRISPR CRISPR-Cas Systems Deoxyribonucleic acid DNA DNA Barcoding, Taxonomic - methods DNA sequencing DNA, Fungal - genetics Editing Fusion protein Gene Editing - methods Genetic aspects Genetic diversity Genetic variance Genome editing Genome, Fungal Genomes Genotypes Homologous Recombination Life Sciences Methods Multiplexing Organic chemistry Phenotypes Phenotyping Phospholipid Transfer Proteins - genetics Plasmids - genetics Proteins RNA, Fungal - genetics Saccharomyces cerevisiae Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae Proteins - genetics Yeasts Yeasts (Fungi)
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Summary:	A method to introduce defined mutations into the yeast genome enables saturation mutagenesis of a gene and genome-scale introduction of genetic variants. Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess the phenotypic consequences of each perturbation. Here we describe a CRISPR–Cas9-based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) in Saccharomyces cerevisiae. MAGESTIC uses array-synthesized guide–donor oligos for plasmid-based high-throughput editing and features genomic barcode integration to prevent plasmid barcode loss and to enable robust phenotyping. We demonstrate that editing efficiency can be increased more than fivefold by recruiting donor DNA to the site of breaks using the LexA–Fkh1p fusion protein. We performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. We also constructed thousands of natural genetic variants, characterized guide mismatch tolerance at the genome scale, and ascertained that cryptic Pol III termination elements substantially reduce guide efficacy. MAGESTIC will be broadly useful to uncover the genetic basis of phenotypes in yeast.
Bibliography:	These authors contributed equally to this work
ISSN:	1087-0156 1546-1696
DOI:	10.1038/nbt.4137